Method for producing RNA compositions

ABSTRACT

The present invention relates to a method for producing a liquid composition comprising a nanoparticle comprising at least one RNA and at least one cationic or polycationic compound, advantageously on a large scale suitable for pharmaceutical applications. The present invention further concerns the use of the inventive method in the manufacture of a medicament or a vaccine. Furthermore, the invention relates to compositions containing the RNA-comprising nanoparticle, and to pharmaceutical compositions comprising the same.

This application is a continuation of U.S. application Ser. No.15/566,010, filed Oct. 12, 2017, now U.S. Pat. No. 10,653,768, which isa national phase application under 35 U.S.C. § 371 of InternationalApplication No. PCT/EP2016/000607, filed Apr. 13, 2016, which claimsbenefit of International Application No. PCT/EP2015/000771, filed Apr.13, 2015, the entire contents of each of which are hereby incorporatedby reference.

The sequence listing that is contained in the file named“CRVCP0172USC1.txt”, which is 5 KB (as measured in Microsoft Windows®)and was created on Apr. 1, 2020, is filed herewith by electronicsubmission and is incorporated by reference herein.

The present invention relates to a method for producing a liquidcomposition comprising a nanoparticle comprising at least one RNA and atleast one cationic or polycationic compound, advantageously on a largescale suitable for pharmaceutical applications. The present inventionfurther concerns the use of the inventive method in the manufacture of amedicament or a vaccine. Furthermore, the invention relates tocompositions containing the RNA-comprising nanoparticle, and topharmaceutical compositions comprising the same.

Therapeutic ribonucleic acid (RNA) molecules represent an emerging classof drugs. RNA-based therapeutics include mRNA molecules encodingantigens for use as vaccines (Fotin-Mleczek et al. 2012. J. Gene Med.14(6):428-439). In addition, it is envisioned to use RNA molecules forreplacement therapies, e.g. providing missing proteins such as growthfactors or enzymes to patients (Karikó et al., 2012. Mol. Ther.20(5):948-953; Kormann et al., 2012. Nat. Biotechnol. 29(2):154-157).Furthermore, the therapeutic use of noncoding immunostimulatory RNAmolecules (cf. e.g. WO 2009/095226 A2) and other noncoding RNAs such asmicroRNAs and long noncoding RNAs is considered (Esteller, 2011. Nat.Rev. Genet. 12(12):861-74).

It has been found that the successful in vivo delivery of nucleic acidsincluding RNA depends on the formulation of the active molecules intodosage forms suitable for the therapeutic application. In this context,particularly, the complexation of nucleic acids with polycatoniccompounds, which results in nanoparticles, has been found to improve thein vivo delivery of nucleic acids, especially of RNA.

Different nucleic acid-comprising nanoparticles and methods for thepreparation thereof have been described in the art, for example in thedocuments listed in the following.

WO 2010/037539 A1 describes immunostimulatory compositions comprising a)an adjuvant component, comprising or consisting of at least one (m)RNA,complexed with a cationic or polycationic compound, and b) at least onefree mRNA, encoding at least one therapeutically active protein,antigen, allergen and/or antibody, wherein the immunostimulatorycomposition is capable to elicit or enhance an innate and optionally anadaptive immune response in a mammal.

WO 2009/144230 A1 describes protamine/RNA-comprising nanoparticles ofdefined average size, a pharmaceutical composition containing saidnanoparticles and to a method of producing the same. The production ofnanoparticles from RNA and protamine is described to comprise mixingconducted by pipette or vortexing.

WO 2012/094574 describes PEG-6-PPA/siRNA comprising nanoparticles ofdefined average particle size, and a pharmaceutical compositioncontaining said nanoparticles and a method of producing the same. Theproduction of nanoparticles from siRNA is described to comprise mixingconducted by pipette or vortexing. This document teaches that the timeto allow for complexation is between 5 minutes to 60 minutes.

Yang and colleagues (Liu, Zhenzhen, et al., International journal ofnanomedicine 10 (2015): 2735) describe a liquid composition comprisingnanoparticles comprising siRNA and protamine. The production ofnanoparticles from siRNA was conducted by vortexing of the twocomponents (2.5 μl siRNA and protamine) and subsequent incubation for 20minutes at room temperature.

In the above mentioned prior art, complexation of the nucleic acid (inparticular of RNA) with cationic or polycationic compounds is alwaysobtained by pipetting or vortexing. Typically, only relatively smallvolumes (<1 mL) are prepared on a laboratory scale by simply adding RNAto a solution of cationic or polycationic compounds (or vice versa) withsubsequent mixing by repeated inversion or pipetting. Under suchcircumstances, it is virtually impossible to standardize the reactionconditions, which results in the variability of the physicalcharacteristics of the produced nanoparticles. Such variability isfurther exacerbated by attempts to scale-up reaction volumes forpre-clinical or clinical studies, and further market delivery, wheredifferences in physical characteristics are not acceptable. Therefore,these methods are not applicable for the large-scale production ofRNA-comprising nanoparticles for medical purposes because they cannotwarrant a constant process, which results in uniform batches ofRNA-comprising nanoparticles.

Further methods and devices to produce nucleic acid-comprisingnanoparticles are known from the prior art.

For example, a pneumatic mixing device that enables the production oflarge volumes of nonviral gene therapy formulations was reported (Davieset al., 2010. Biotechniques 49(3):666-668). This mixing device usescompressed air to depress the plunger of a disposable dual barrelpolypropylene syringe containing plasmid DNA and lipid/polymer inseparate compartments. Activation of the device initiates mixing of thecomponents by simultaneous extrusion of the two reagents through astatic mixer device.

WO 1999/040771 describes concurrent flow mixing methods and apparatusesusing e.g. static or dynamic mixers that could be adapted for thepreparation of gene therapy vector and vehicle compositions ofcontrolled particle size for condensate complexes. However, actualtesting was only carried out on a microliter scale

WO 2009/039657 A1 describes highly concentrated chitosan-nucleic acidpolyplex compositions and dispersions. Methods of mixing thechitosan-nucleic acid polyplexes include an inline mixing of chitosansolution and nucleic acid solution, followed by further concentratingthe dispersion of chitosan-nucleic acid polyplexes.

A method for the preparation of siRNA-containing lipid nanoparticels bycontrolled microfluidic formulation was described by Chen et al., J. Am.Chem. Soc. 2012. 134(16):6948-6951. The formulation method is based onstepwise ethanol dilution to produce siRNA lipid nanoparticles on amicroliter scale.

Zhang et al. 2012 (Polydispersity characterization of lipidnanoparticles for siRNA delivery using multiple detection size-exclusionchromatography. Anal Chem. 84(14):6088-96) describe that the developmentof lipid nanoparticle (LNP) based small interfering RNA (siRNA)therapeutics presents unique pharmaceutical and regulatory challenges.In contrast to small molecule drugs that are highly pure andwell-defined, LNP drug products can exhibit heterogeneity in size,composition, surface property, or morphology. The potential for batchheterogeneity introduces a complexity that must be addressed in order tosuccessfully develop and ensure quality in LNP pharmaceuticals. Despitethe similarity in the particle assembly process, it was found here thatone LNP batch possessed a narrow particle size and molecular weightdistribution while the other was polydisperse. These results suggestthat LNP drug products are highly complex and diverse in nature, andcare should be taken in examining and understanding them to ensure theirquality and consistency. The authors concluded that currently there is alack of scientific knowledge concerning heterogeneity of LNPs as well ashigh-resolution techniques that permit its evaluation.

In summary, all conventional bulk techniques for the preparation ofnucleic acid (RNA) formulations, such as RNA-comprising nanoparticles,which involve uncontrolled mixing of the compound solution and the RNAsolution (or vice versa), may face limitations including poorreproducibility, polydisperse particle size distribution andbatch-to-batch variation with respect to physicochemical properties ofthe nanoparticle, especially when performed at a large scale. Moreover,as RNA therapeutics are applied for various different indications,different batch sizes are needed and thus, flexibly scalable methods forthe preparation of RNA-comprising nanoparticle formulations aresought-after.

In view of the above-mentioned prior art, there is a continued need forimproved, economical and flexibly scalable means and methods ofproducing RNA-comprising nanoparticles, especially for the large scalepreparation of RNA therapeutics. Particularly, there is a need toproduce uniform large-scale batches of RNA-comprising nanoparticleshaving similar average particles sizes and polydispersity. Especially,for pre-clinical and clinical studies, and eventually the marketdelivery of a medicament based on a RNA-comprising compound and/ornanoparticles, a production method is required to allow a reproducibleproduction of RNA-comprising compound and/or nanoparticles, which are tobe obtained with a reliable quality (i.e. constant physicalcharacteristics) in the large scale production thereof.

In view of the above-discussed problem, the inventors of the presentinvention conducted intensive studies with regard to up-scaling of theproduction of RNA-comprising nanoparticles suitable for pharmaceuticalapplications, especially RNA-comprising nanoparticles, which arecharacterised by having uniform average particles sizes andpolydispersity. The inventors have surprisingly found that, independentof reagents and scale, a reliable production of a uniform RNA-comprisingcompound can be obtained in a reproducible manner, if thecompound-forming reaction is conducted using a reactor with a blend timeof 5 seconds or less. More preferably, the blend time is 2.5 seconds orless, 2.0 seconds or less, 1.0 seconds or less, or 0.5 seconds or less.Alternatively, the blend time is preferably in a range from about 0.001seconds to about 5 seconds, more preferably from about 0.01 seconds toabout 5 seconds, even more preferably from about 0.1 seconds to about 5seconds and most preferably from about 0.001 to about 2 seconds or fromabout 0.01 to about 2 seconds.

In light of the above described prior art, the inventors unexpectedlyfound that decreasing the blend time of the compound-forming reactionincreases the homogeneity of the compound-containing mixture and alsoincreases the uniformity of the nanoparticles.

Moreover, the inventors have surprisingly found that the so-obtainedcompositions comprising RNA-comprising compounds exhibit a very highquality of uniform RNA-comprising compound in high yields, withoutcomprising significant amounts of unwanted side products, which usuallylead to problems concerning the stability and/or applicability of theproduct compositions. In particular, a liquid composition can beobtained by the method according to the present invention, wherein RNAis present in a complex with a cationic or polycationic compound, whileno undesired precipitates or aggregates are formed.

On basis of these findings, the inventors have completed the presentinvention, which provides a reliable method for producing aRNA-comprising nanoparticle in a quality sufficient and reliable forpharmaceutical applications, even in a large scale production. Further,the invention provides a production process both cost-effective andreliable on a large scale. The present invention was made with supportfrom the Government under Agreement No. HR0011-11-3-0001 awarded byDARPA. The Government has certain rights in the invention.

In particular, the present invention provides a method for producing aliquid composition comprising a nanoparticle comprising at least one RNAand at least one cationic or polycationic compound,

wherein the method comprises the steps of:

(a) providing a first liquid composition comprising at least one RNA,

(b) providing a second liquid composition comprising at least onecationic or polycationic compound,

(c) introducing the first liquid composition and the second liquidcomposition into at least one reactor, wherein the at least one RNA andthe at least one cationic or polycationic compound are mixed with ablend time of 5 seconds or less and reacted with each other, and

(d) recovering the product liquid composition comprising thenanoparticle comprising the at least one RNA and the at least onecationic or polycationic compound from the reactor.

It was found that the method according to the present invention producesa nanoparticle comprising at least one RNA and at least one cationic orpolycationic compound, reliably under controlled conditions, withoutallowing unwanted side reactions resulting in unwanted side products andstability and/or applicability problems caused thereby, independent ofthe scale of production. Further, the method according to the presentinvention is both cost-effective and reliable, even on a large scale,which renders the method of the invention especially suitable for thepharmaceutical production of RNA-comprising nanoparticles. Preferablyand advantageously, the method according to the invention is used forthe production of a nanoparticle comprising at least one RNA and atleast one cationic or polycationic compound on a large scale, preferablyan industrial scale of pharmaceutical production.

In the context of the invention, the term “large scale” (sometimes“large scale batch”) refers to an amount of RNA-comprising compound,which summarily comprises RNA in an amount of 1 g or more, preferably inan amount of 5 g and more, and even more preferred in an amount of 10 gor more.

In the following description of the present invention and its preferredembodiments, if not otherwise indicated, different features ofalternatives and embodiments may be combined with each other, wheresuitable. Furthermore, the term “comprising” shall not be construed asmeaning “consisting of”, if not specifically mentioned. However, in thecontext of the present invention, term “comprising” may be substitutedwith the term “consisting of”, where suitable.

In the context of the invention, the term RNA is used to indicate anytype of ribonucleic acid.

Examples of RNA, which can be used in the method of the presentinvention are disclosed, e.g. in WO 2008/077592 A1, WO 2009/095226 A2,WO 2010/037539 and WO 2011/026641 A1, which are all incorporated hereinby reference.

Preferably, the at least one RNA is selected from the group consistingof a long-chain RNA, a coding RNA, a non-coding RNA, a messenger RNA(mRNA), an RNA oligonucleotide, an siRNA, an shRNA, an antisense RNA, ariboswitch, an immunostimulating RNA (isRNA), a ribozyme or an aptamer;etc. The RNA may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA),a messenger RNA (mRNA), a viral RNA (vRNA) or a replicon RNA.Preferably, the RNA is a coding RNA. Even more preferably, the RNA is a(linear) single-stranded RNA, even more preferably an mRNA.

Alternatively, the at least one RNA may be selected from the groupconsisting of a long-chain RNA, a coding RNA, a non-coding RNA, a singlestranded RNA (ssRNA), a double stranded RNA (dsRNA), a linear RNA(linRNA), a circular RNA (circRNA), a messenger RNA (mRNA), an RNAoligonucleotide, a small interfering RNA (siRNA), a small hairpin RNA(shRNA), an antisense RNA (asRNA), a CRISPR/Cas9 guide RNA, ariboswitch, an immunostimulating RNA (isRNA), a ribozyme or an aptamer;etc. The RNA may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA),a messenger RNA (mRNA), a viral RNA (vRNA), a retroviral RNA, or areplicon RNA, a small nuclear RNA (snRNA), a small nucleolar RNA(snoRNA), a microRNA (miRNA), and a Piwi-interacting RNA (piRNA).Preferably, the RNA is a coding RNA.

In the context of the present invention, an mRNA is typically an RNA,which is composed of several structural elements, e.g. an optional5′-UTR region, an upstream positioned ribosomal binding site followed bya coding region, an optional 3′-UTR region, which may be followed by apoly-A tail (and/or a poly-C-tail). An mRNA may occur as a mono-, di-,or even multicistronic RNA, i.e. an RNA which carries the codingsequences of one, two or more (identical or different) proteins orpeptides as defined herein. Such coding sequences in di-, or evenmulticistronic mRNA may be separated by at least one IRES (internalribosomal entry site) sequence.

Furthermore, the at least one RNA may be a single- or a double-strandedRNA (molecule) or a partially double-stranded or partially singlestranded RNA, which are at least partially self complementary (both ofthese partially double-stranded or partially single stranded RNAmolecules are typically formed by a longer and a shorter single-strandedRNA molecule or by two single stranded RNA molecules, which are aboutequal in length, wherein one single-stranded RNA molecule is in partcomplementary to the other single-stranded RNA molecule and both thusform a double-stranded nucleic RNA in this region, i.e. a partiallydouble-stranded or partially single stranded RNA (molecule). Preferably,the at least one RNA is a single-stranded RNA molecule. Furthermore, theRNA (molecule) may be a circular or linear RNA molecule, preferably alinear RNA molecule.

Coding RNA:

The at least one RNA may encode a protein or a peptide, which may beselected, without being restricted thereto, e.g. from therapeuticallyactive proteins or peptides, selected e,g, from adjuvant proteins, fromantigens, e.g. tumour antigens, pathogenic antigens (e.g. selected, fromanimal antigens, from viral antigens, from protozoal antigens, frombacterial antigens), allergenic antigens, autoimmune antigens, orfurther antigens, from allergens, from antibodies, fromimmunostimulatory proteins or peptides, from antigen-specific T-cellreceptors, or from any other protein or peptide suitable for a specific(therapeutic) application, wherein the coding RNA may be transportedinto a cell, a tissue or an organism and the protein may be expressedsubsequently in this cell, tissue or organism.

The coding region of the at least one RNA may occur as a mono-, di-, oreven multicistronic RNA, i.e. an RNA, which carries the coding sequencesof one, two or more proteins or peptides. Such coding sequences in di-,or even multicistronic RNA may be separated by at least one internalribosome entry site (IRES) sequence, or by signal peptides which inducethe cleavage of the resulting polypeptide, which comprises severalproteins or peptides.

In particular preferred embodiments, the encoded peptides or proteinsare selected from human, viral, bacterial, protozoan proteins orpeptides.

a) Therapeutically Active Proteins

-   -   In the context of the present invention, therapeutically active        proteins or peptides may be encoded by the at least one RNA.        Therapeutically active proteins are defined herein as proteins,        which have an effect on healing, prevent prophylactically or        treat therapeutically a disease, preferably as defined herein,        or are proteins of which an individual is in need of. These may        be selected from any naturally or synthetically designed        occurring recombinant or isolated protein known to a skilled        person from the prior art. Without being restricted thereto        therapeutically active proteins may comprise proteins, capable        of stimulating or inhibiting the signal transduction in the        cell, e.g. cytokines, lymphokines, monokines, growth factors,        receptors, signal transduction molecules, transcription factors,        etc; anticoagulants; antithrombins; antiallergic proteins;        apoptotic factors or apoptosis related proteins, therapeutic        active enzymes and any protein connected with any acquired        disease or any hereditary disease.

A therapeutically active protein, which may be encoded by the RNA, mayalso be an adjuvant protein. In this context, an adjuvant protein ispreferably to be understood as any protein, which is capable to elicitan innate immune response as defined herein. Preferably, such an innateimmune response comprises activation of a pattern recognition receptor,such as e.g. a receptor selected from the Toll-like receptor (TLR)family, including e.g. a Toll like receptor selected from human TLR1 toTLR10 or from murine Toll like receptors TLR1 to TLR13. More preferably,the adjuvant protein is selected from human adjuvant proteins or frompathogenic adjuvant proteins, selected from the group consisting of,without being limited thereto, bacterial proteins, protozoan proteins,viral proteins, or fungal proteins, animal proteins, in particular frombacterial adjuvant proteins. In addition, RNA encoding human proteinsinvolved in adjuvant effects (e.g. ligands of pattern recognitionreceptors, pattern recoginition receptors, proteins of the signaltransduction pathways, transcription factors or cytokines) may be usedas well.

b) Antigens

-   -   The at least one RNA may alternatively encode an antigen.        According to the present invention, the term “antigen” refers to        a substance, which is recognized by the immune system and is        capable of triggering an antigen-specific immune response, e.g.        by formation of antibodies or antigen-specific T-cells as part        of an adaptive immune response. In this context an antigenic        epitope, fragment or peptide of a protein means particularly B        cell and T cell epitopes, which may be recognized by B cells,        antibodies or T cells respectively.    -   In the context of the present invention, antigens as encoded by        the at least one RNA typically comprise any antigen, antigenic        epitope or antigenic peptide, falling under the above        definition, more preferably protein and peptide antigens, e.g.        tumour antigens, allergenic antigens, auto-immune self-antigens,        pathogenic antigens, etc. In particular antigens as encoded by        the RNA may be antigens generated outside the cell, more        typically antigens not derived from the host organism (e.g. a        human) itself (i.e. non-self antigens) but rather derived from        host cells outside the host organism, e.g. viral antigens,        bacterial antigens, fungal antigens, protozoological antigens,        animal antigens, allergenic antigens, etc. Allergenic antigens        (allergy antigens) are typically antigens, which cause an        allergy in a human and may be derived from either a human or        other sources. Additionally, antigens as encoded by the RNA may        be furthermore antigens generated inside the cell, the tissue or        the body. Such antigens include antigens derived from the host        organism (e.g. a human) itself, e.g. tumour antigens,        self-antigens or auto-antigens, such as auto-immune        self-antigens, etc., but also (non-self) antigens as defined        herein, which have been originally been derived from host cells        outside the host organism, but which are fragmented or degraded        inside the body, tissue or cell, e.g. by (protease) degradation,        metabolism, etc.    -   One class of antigens as encoded by the RNA comprises tumour        antigens. “Tumour antigens” are preferably located on the        surface of the (tumour) cell. Tumour antigens may also be        selected from proteins, which are overexpressed in tumour cells        compared to a normal cell. Furthermore, tumour antigens also        include antigens expressed in cells, which are (were) not        themselves (or originally not themselves) degenerated but are        associated with the supposed tumour. Antigens, which are        connected with tumour-supplying vessels or (re)formation        thereof, in particular those antigens, which are associated with        neovascularization, e.g. growth factors, such as VEGF, bFGF        etc., are also included herein. Antigens connected with a tumour        furthermore include antigens from cells or tissues, typically        embedding the tumour. Further, some substances (usually proteins        or peptides) are expressed in patients suffering (knowingly or        not-knowingly) from a cancer disease and they occur in increased        concentrations in the body fluids of said patients. These        substances are also referred to as “tumour antigens”, however        they are not antigens in the stringent meaning of an immune        response inducing substance. The class of tumour antigens can be        divided further into tumour-specific antigens (TSAs) and        tumour-associated-antigens (TAAs). TSAs can only be presented by        tumour cells and never by normal “healthy” cells. They typically        result from a tumour specific mutation. TAAs, which are more        common, are usually presented by both tumour and healthy cells.        These antigens are recognized and the antigen-presenting cell        can be destroyed by cytotoxic T cells. Additionally, tumour        antigens can also occur on the surface of the tumour in the form        of, e.g., a mutated receptor. In this case, they can be        recognized by antibodies.    -   According to another alternative, one further class of antigens        as encoded by the RNA comprises allergenic antigens. Such        allergenic antigens may be selected from antigens derived from        different sources, e.g. from animals, plants, fungi, bacteria,        etc. Allergens in this context include e.g. grasses, pollens,        molds, drugs, or numerous environmental triggers, etc.

c) Antibodies

-   -   According to a further alternative, the RNA may encode an        antibody or an antibody fragment. According to the present        invention, such an antibody may be selected from any antibody,        e.g. any recombinantly produced or naturally occurring        antibodies, known in the art, in particular antibodies suitable        for therapeutic, diagnostic or scientific purposes, or        antibodies, which have been identified in relation to specific        cancer diseases. Herein, the term “antibody” is used in its        broadest sense and specifically covers monoclonal and polyclonal        antibodies (including agonist, antagonist, and blocking or        neutralizing antibodies) and antibody species with polyepitopic        specificity. According to the invention, the term “antibody”        typically comprises any antibody known in the art (e.g. IgM,        IgD, IgG, IgA and IgE antibodies), such as naturally occurring        antibodies, antibodies generated by immunization in a host        organism, antibodies which were isolated and identified from        naturally occurring antibodies or antibodies generated by        immunization in a host organism and recombinantly produced by        biomolecular methods known in the art, as well as chimeric        antibodies, human antibodies, humanized antibodies, bispecific        antibodies, intrabodies, i.e. antibodies expressed in cells and        optionally localized in specific cell compartments, and        fragments and variants of the aforementioned antibodies. In        general, an antibody consists of a light chain and a heavy chain        both having variable and constant domains. The light chain        consists of an N-terminal variable domain, V_(L), and a        C-terminal constant domain, C_(L). In contrast, the heavy chain        of the IgG antibody, for example, is comprised of an N-terminal        variable domain, V_(H), and three constant domains,

C_(H)1, C_(H)2 and C_(H)3.

-   -   In the context of the present invention, antibodies as encoded        by the at least one RNA may preferably comprise full-length        antibodies, i.e. antibodies composed of the full heavy and full        light chains, as described above. However, derivatives of        antibodies such as antibody fragments, variants or adducts may        also be encoded by the RNA. Antibody fragments are preferably        selected from Fab, Fab′, F(ab′)₂, Fc, Facb, pFc′, Fd and Fv        fragments of the aforementioned (full-length) antibodies. In        general, antibody fragments are known in the art. For example, a        Fab (“fragment, antigen binding”) fragment is composed of one        constant and one variable domain of each of the heavy and the        light chain. The two variable domains bind the epitope on        specific antigens. The two chains are connected via a disulfide        linkage. A scFv (“single chain variable fragment”) fragment, for        example, typically consists of the variable domains of the light        and heavy chains. The domains are linked by an artificial        linkage, in general a polypeptide linkage such as a peptide        composed of 15-25 glycine, proline and/or serine residues.

In the present context, it is preferable that the different chains ofthe antibody or antibody fragment are encoded by a multicistronic RNA.Alternatively, the different strains of the antibody or antibodyfragment are encoded by several monocistronic RNA (sequences).

siRNA:

According to a further alternative, the at least one RNA may be in theform of dsRNA, preferably siRNA. A dsRNA, or a siRNA, is of interestparticularly in connection with the phenomenon of RNA interference. Thein vitro technique of RNA interference (RNAi) is based ondouble-stranded RNA molecules (dsRNA), which trigger thesequence-specific suppression of gene expression (Zamore (2001) Nat.Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5:485-490: Hannon(2002) Nature 41: 244-251). In the transfection of mammalian cells withlong dsRNA, the activation of protein kinase R and RnaseL brings aboutunspecific effects, such as, for example, an interferon response (Starket al. (1998) Annu. Rev. Biochem. 67: 227-264; He and Katze (2002) ViralImmunol. 15: 95-119). These unspecific effects are avoided when shorter,for example 21- to 23-mer, so-called siRNA (small interfering RNA), isused, because unspecific effects are not triggered by siRNA that isshorter than 30 bp (Elbashir et al. (2001) Nature 411: 494-498).

The RNA may thus be a double-stranded RNA (dsRNA) having a length offrom 17 to 29, preferably from 19 to 25, and preferably being at least90%, more preferably 95% and especially 100% (of the nucleotides of adsRNA) complementary to a section of the RNA sequence of a(therapeutically relevant) protein or antigen described (as activeingredient) hereinbefore, either a coding or a non-coding section,preferably a coding section. 90% complementary means that with a lengthof a dsRNA described herein of, for example, 20 nucleotides, thiscontains not more than 2 nucleotides without correspondingcomplementarity with the corresponding section of the mRNA. The sequenceof the double-stranded RNA is, however, preferably wholly complementaryin its general structure with a section of the RNA of a therapeuticallyrelevant protein or antigen described hereinbefore. In this context theRNA may be a dsRNA having the general structure 5′-(N₁₇₋₂₃)-3′,preferably having the general structure 5′-(N₁₃₋₂₅)-3′, more preferablyhaving the general structure 5′-(N₁₋₂₄)-3′, or yet more preferablyhaving the general structure 5′-(N₂₁₋₂₃)-3′, wherein for each generalstructure each N is a (preferably different) nucleotide of a section ofthe mRNA of a therapeutically relevant protein or antigen describedhereinbefore, preferably being selected from a continuous number of 17to 29 nucleotides of the mRNA of a therapeutically relevant protein orantigen and being present in the general structure 5′-(N₁₇₋₂₃)-3′ intheir natural order. In principle, all the sections having a length offrom 17 to 29, preferably from 19 to 25, base pairs that occur in thecoding region of the mRNA can serve as target sequence for a dsRNAherein. Equally, dsRNAs used as RNA can also be directed againstnucleotide sequences of a (therapeutically relevant) protein or antigendescribed (as active ingredient) hereinbefore that do not lie in thecoding region, in particular in the 5′ non-coding region of the mRNA,for example, therefore, against non-coding regions of the mRNA having aregulatory function. The target sequence of the dsRNA used as RNA cantherefore lie in the translated and untranslated region of the mRNAand/or in the region of the control elements of a protein or antigendescribed hereinbefore. The target sequence of a dsRNA used as RNA canalso lie in the overlapping region of untranslated and translatedsequence; in particular, the target sequence can comprise at least onenucleotide upstream of the start triplet of the coding region of themRNA.

Immunostimulatory RNA:

a) Immunostimulatory CpG Nucleic Acids:

-   -   According to another alternative, the at least one RNA may be in        the form of a a(n) (immunostimulatory) CpG-RNA, which preferably        induces an innate immune response. A CpG-RNA can be a        single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded        CpG-RNA (ds CpG-RNA). The CpG-RNA is preferably in the form of        single-stranded CpG-RNA (ss CpG-RNA). Also preferably, such CpG        RNA have a length as described above. Preferably, the CpG motifs        are unmethylated.

b) Immunostimulatory RNA (isRNA):

-   -   Likewise, according to a further alternative, the at least one        RNA may be in the form of an immunostimulatory RNA (isRNA),        which preferably elicits an innate immune response. Such an        immunostimulatory RNA may be any (double-stranded or        single-stranded) RNA, e.g. a coding RNA, as defined herein.        Preferably, the immunostimulatory RNA may be a single-stranded,        a double-stranded or a partially double-stranded RNA, more        preferably a single-stranded RNA, and/or a circular or linear        RNA, more preferably a linear RNA. More preferably, the        immunostimulatory RNA may be a (linear) single-stranded RNA.        Even more preferably, the immunostimulatory RNA may be a (long)        (linear) single-stranded) non-coding RNA. In this context it is        particular preferred that the isRNA carries a triphosphate at        its 5′-end which is the case for in vitro transcribed RNA. An        immunostimulatory RNA may also occur as a short RNA        oligonucleotide as defined herein. An immunostimulatory RNA as        used herein may furthermore be selected from any class of RNA        molecules, found in nature or being prepared synthetically, and        which can induce an innate immune response and may support an        adaptive immune response induced by an antigen. In this context,        an immune response may occur in various ways. A substantial        factor for a suitable (adaptive) immune response is the        stimulation of different T-cell sub-populations. T-lymphocytes        are typically divided into two sub-populations, the T-helper 1        (Th1) cells and the T-helper 2 (Th2) cells, with which the        immune system is capable of destroying intracellular (Th1) and        extracellular (Th2) pathogens (e.g. antigens). The two Th cell        populations differ in the pattern of the effector proteins        (cytokines) produced by them. Thus, Th1 cells assist the        cellular immune response by activation of macrophages and        cytotoxic T-cells. Th2 cells, on the other hand, promote the        humoral immune response by stimulation of B-cells for conversion        into plasma cells and by formation of antibodies (e.g. against        antigens). The Th1/Th2 ratio is therefore of great importance in        the induction and maintenance of an adaptive immune response. In        connection with the present invention, the Th1/Th2 ratio of the        (adaptive) immune response is preferably shifted in the        direction towards the cellular response (Th1 response) and a        cellular immune response is thereby induced. According to one        example, the innate immune system which may support an adaptive        immune response, may be activated by ligands of Toll-like        receptors (TLRs). TLRs are a family of highly conserved pattern        recognition receptor (PRR) polypeptides that recognize        pathogen-associated molecular patterns (PAMPs) and play a        critical role in innate immunity in mammals. Currently at least        thirteen family members, designated TLR1-TLR13 (Toll-like        receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,        TLR10, TLR11, TLR12 or TLR13), have been identified.        Furthermore, a number of specific TLR ligands have been        identified. It was e.g. found that unmethylated bacterial DNA        and synthetic analogs thereof (CpG DNA) are ligands for TLR9        (Hemmi H et al. (2000) Nature 408:740-5; Bauer S et al. (2001)        Proc NatlAcadSci USA 98, 9237-42). Furthermore, it has been        reported that ligands for certain TLRs include certain nucleic        acid molecules and that certain types of RNA are        immunostimulatory in a sequence-independent or        sequence-dependent manner, wherein these various        immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8,        or intracellular receptors such as RIG-I, MDA-5, etc. E.g.        Lipford et al. determined certain G,U-containing        oligoribonucleotides as immunostimulatory by acting via TLR7 and        TLR8 (see WO 03/086280). The immunostimulatory G,U-containing        oligoribonucleotides described by Lipford et al. were believed        to be derivable from RNA sources including ribosomal RNA,        transfer RNA, messenger RNA, and viral RNA.

The immunostimulatory RNA (isRNA) may thus comprise any RNA sequenceknown to be immunostimulatory, including, without being limited thereto,RNA sequences representing and/or encoding ligands of TLRs, preferablyselected from human family members TLR1-TLR10 or murine family membersTLR1-TLR13, more preferably selected from (human) family membersTLR1-TLR10, even more preferably from TLR7 and TLR8, ligands forintracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see e.g.Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases:two parallel ways to trigger antiviral responses. Mol. Cell 22,561-569), or any other immunostimulatory RNA sequence. Furthermore,(classes of) immunostimulatory RNA molecules may include any other RNAcapable of eliciting an immune response. Without being limited thereto,such an immunostimulatory RNA may include ribosomal RNA (rRNA), transferRNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA). Such animmunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of5 to 50 or of 5 to 30 nucleotides.

According to a particularly preferred embodiment of the presentinvention, such immunostimulatory RNA consist of or comprise an RNA offormula (I) or (II):G_(l)X_(m)G_(n)  (formula (I)),

-   -   wherein:    -   G is guanosine, uracil or an analogue of guanosine or uracil;    -   X is guanosine, uracil, adenosine, thymidine, cytosine or an        analogue of the above-mentioned nucleotides;    -   l is an integer from 1 to 40,        -   wherein        -   when l=1 G is guanosine or an analogue thereof,        -   when l>1 at least 50% of the nucleotides are guanosine or an            analogue thereof;    -   m is an integer and is at least 3;        -   wherein        -   when m=3 X is uracil or an analogue thereof,        -   when m>3 at least 3 successive uracils or analogues of            uracil occur;    -   n is an integer from 1 to 40,        -   wherein        -   when n=1 G is guanosine or an analogue thereof,        -   when n>1 at least 50% of the nucleotides are guanosine or an            analogue thereof.            C_(l)X_(m)C_(n)  (formula (II)),    -   wherein:    -   C is cytosine, uracil or an analogue of cytosine or uracil;    -   X is guanosine, uracil, adenosine, thymidine, cytosine or an        analogue of the above-mentioned nucleotides;    -   l is an integer from 1 to 40,        -   wherein        -   when l=1 C is cytosine or an analogue thereof,        -   when l>1 at least 50% of the nucleotides are cytosine or an            analogue thereof;    -   m is an integer and is at least 3;        -   wherein        -   when m=3 X is uracil or an analogue thereof,        -   when m>3 at least 3 successive uracils or analogues of            uracil occur;    -   n is an integer from 1 to 40,        -   wherein        -   when n=1 C is cytosine or an analogue thereof,        -   when n>1 at least 50% of the nucleotides are cytosine or an            analogue thereof.

The RNA of formula (I) or (II) may be relatively short nucleic acidmolecules with a typical length of approximately from 5 to 100 (but mayalso be longer than 100 nucleotides for specific embodiments, e.g. up to200 nucleotides), from 5 to 90 or from 5 to 80 nucleotides, preferably alength of approximately from 5 to 70, more preferably a length ofapproximately from 8 to 60 and, more preferably a length ofapproximately from 15 to 60 nucleotides, more preferably from 20 to 60,most preferably from 30 to 60 nucleotides. If the RNA has a maximumlength of e.g. 100 nucleotides, m will typically be <=98. The number ofnucleotides G in the RNA of formula (I) is determined by l or n. l andn, independently of one another, are each an integer from 1 to 40,wherein when l or n=1 G is guanosine or an analogue thereof, and when lor n>1 at least 50% of the nucleotides are guanosine or an analoguethereof. A nucleotide adjacent to X, in the RNA of formula (II)according to the invention is preferably not a uracil. Preferably, forformula (I), when l or n>1, at least 60%, 70%, 80%, 90% or even 100% ofthe nucleotides are guanosine or an analogue thereof, as defined above.The remaining nucleotides to 100% (when guanosine constitutes less than100% of the nucleotides) in the flanking sequences G₁ and/or G_(n) areuracil or an analogue thereof, as defined hereinbefore. Also preferably,l and n, independently of one another, are each an integer from 2 to 30,more preferably an integer from 2 to 20 and yet more preferably aninteger from 2 to 15. The lower limit of l or n can be varied ifnecessary and is at least 1, preferably at least 2, more preferably atleast 3, 4, 5, 6, 7, 8, 9 or 10. This definition applies correspondinglyto formula (IV).

According to a further particularly preferred embodiment, suchimmunostimulatory RNA, consists of or comprises an RNA of formula (III)or (IV):(N_(u)G_(l)X_(m)G_(n)N_(v))_(a)  (formula (III)),

wherein:

-   -   G is guanosine (guanine), uridine (uracil) or an analogue of        guanosine (guanine) or uridine (uracil), preferably guanosine        (guanine) or an analogue thereof;    -   X is guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine), or an analogue of        these nucleotides (nucleosides), preferably uridine (uracil) or        an analogue thereof;    -   N is a nucleic acid sequence having a length of about 4 to 50,        preferably of about 4 to 40, more preferably of about 4 to 30 or        4 to 20 nucleic acids, each N independently being selected from        guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine) or an analogue of these        nucleotides (nucleosides);    -   a is an integer from 1 to 20, preferably from 1 to 15, most        preferably from 1 to 10;    -   l is an integer from 1 to 40,        -   wherein when l=1, G is guanosine (guanine) or an analogue            thereof,            -   when l>1, at least 50% of these nucleotides                (nucleosides) are guanosine (guanine) or an analogue            -   thereof;    -   m is an integer and is at least 3;        -   wherein when m=3, X is uridine (uracil) or an analogue            thereof, and            -   when m>3, at least 3 successive uridines (uracils) or                analogues of uridine (uracil) occur;    -   n is an integer from 1 to 40,        -   wherein when n=1, G is guanosine (guanine) or an analogue            thereof,            -   when n>1, at least 50% of these nucleotides                (nucleosides) are guanosine (guanine) or an analogue            -   thereof;    -   u,v may be independently from each other an integer from 0 to        50,        -   preferably wherein when u=0, v≥1, or            -   when v=0, u≥1;

wherein the RNA molecule of formula (III) has a length of at least 50nucleotides, preferably of at least 100 nucleotides, more preferably ofat least 150 nucleotides, even more preferably of at least 200nucleotides and most preferably of at least 250 nucleotides.(N_(u)C_(l)X_(m)C_(n)N_(v))_(a)  (formula (IV)),

wherein:

-   -   C is cytidine (cytosine), uridine (uracil) or an analogue of        cytidine (cytosine) or uridine (uracil), preferably cytidine        (cytosine) or an analogue thereof;    -   X is guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine) or an analogue of the        above-mentioned nucleotides (nucleosides), preferably uridine        (uracil) or an analogue thereof;    -   N is each a nucleic acid sequence having independent from each        other a length of about 4 to 50, preferably of about 4 to 40,        more preferably of about 4 to 30 or 4 to 20 nucleic acids, each        N independently being selected from guanosine (guanine), uridine        (uracil), adenosine (adenine), thymidine (thymine), cytidine        (cytosine) or an analogue of these nucleotides (nucleosides);    -   a is an integer from 1 to 20, preferably from 1 to 15, most        preferably from 1 to 10;    -   l is an integer from 1 to 40,        -   wherein when l=1, C is cytidine (cytosine) or an analogue            thereof,            -   when l>1, at least 50% of these nucleotides                (nucleosides) are cytidine (cytosine) or an analogue            -   thereof;    -   m is an integer and is at least 3;        -   wherein when m=3, X is uridine (uracil) or an analogue            thereof,            -   when m>3, at least 3 successive uridines (uracils) or                analogues of uridine (uracil) occur;    -   n is an integer from 1 to 40,        -   wherein when n=1, C is cytidine (cytosine) or an analogue            thereof,            -   when n>1, at least 50% of these nucleotides                (nucleosides) are cytidine (cytosine) or an analogue            -   thereof.    -   u, v may be independently from each other an integer from 0 to        50,        -   preferably wherein when u=0, v≥1, or            -   when v=0, u≥1;

wherein the RNA molecule of formula (IV) according to the invention hasa length of at least 50 nucleotides, preferably of at least 100nucleotides, more preferably of at least 150 nucleotides, even morepreferably of at least 200 nucleotides and most preferably of at least250 nucleotides.

For formula (IV), any of the definitions given above for elements N(i.e. N_(u) and N_(v)) and X (X_(m)), particularly the core structure asdefined above, as well as for integers a, l, m, n, u and v, similarlyapply to elements of formula (III) correspondingly, wherein in formula(IV) the core structure is defined by C_(l)X_(m)C_(n). The definition ofbordering elements N_(u) and N_(v) is identical to the definitions givenabove for N_(u) and N_(v).

In a further preferred embodiment the at least one RNA may also occur inthe form of a modified RNA.

According to a further embodiment, the RNA may be provided as a“stabilized RNA”, preferably as a RNA that is essentially resistant toin vivo degradation (e.g. by an exo- or endo-nuclease).

In the context of the present invention, a ‘modified RNA’ is an RNAmolecule comprising at least one modification, preferably as definedherein. In a preferred embodiment, the at least one RNA as used hereincomprises at least one modification as described herein. Preferably, theat least one RNA comprises an RNA modification, which preferablyincreases the stability of the RNA molecule and/or the expression of aprotein encoded by the RNA. Several RNA modifications are known in theart, which can be applied to an RNA molecule in the context of thepresent invention.

Chemical Modifications:

The term “RNA modification” as used herein may refer to chemicalmodifications comprising backbone modifications as well as sugarmodifications or base modifications.

In this context, a modified RNA molecule as defined herein may containnucleotide analogues/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in an RNAmolecule as defined herein are chemically modified. A sugar modificationin connection with the present invention is a chemical modification ofthe sugar of the nucleotides of the RNA molecule as defined herein.Furthermore, a base modification in connection with the presentinvention is a chemical modification of the base moiety of thenucleotides of the RNA molecule. In this context, nucleotide analoguesor modifications are preferably selected from nucleotide analogues,which are applicable for transcription and/or translation.

Sugar Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa RNA molecule as described herein, can be modified in the sugar moiety.For example, the 2′ hydroxyl group (OH) can be modified or replaced witha number of different “oxy” or “deoxy” substituents. Examples of“oxy”-2′ hydroxyl group modifications include, but are not limited to,alkoxy or aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar); polyethyleneglycols (PEG), —O(CH₂CH₂O)nCH₂CH₂OR;“locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected,e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar;and amino groups (—O-amino, wherein the amino group, e.g., NRR, can bealkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) oraminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH2; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA molecule can include nucleotidescontaining, for instance, arabinose as the sugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides, which may be incorporated into a modifiedRNA molecule as described herein. The phosphate groups of the backbonecan be modified by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleosides and nucleotidescan include the full replacement of an unmodified phosphate moiety witha modified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified RNA molecule as described herein can further be modified inthe nucleobase moiety. Examples of nucleobases found in RNA include, butare not limited to, adenine, guanine, cytosine and uracil. For example,the nucleosides and nucleotides described herein can be chemicallymodified on the major groove face. In some embodiments, the major groovechemical modifications can include an amino group, a thiol group, analkyl group, or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-lodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azau ridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(1-Thiophosphate)-Adenosine, 5′-O-(1-Thiophosphate)-Cytidine,5′-O-(1-Thiophosphate)-Guanosine, 5′-O-(1-Thiophosphate)-Uridine or5′-O-(1-Thiophosphate)-Pseudouridine.

In further specific embodiments, a modified RNA may comprise nucleosidemodifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine,7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine,N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine,N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine,7-deaza-adenosine.

Lipid Modification:

According to a further embodiment, a modified RNA molecule as definedherein can contain a lipid modification. Such a lipid-modified RNAmolecule typically comprises an RNA as defined herein. Such alipid-modified RNA molecule as defined herein typically furthercomprises at least one linker covalently linked with that RNA molecule,and at least one lipid covalently linked with the respective linker.Alternatively, the lipid-modified RNA molecule comprises at least oneRNA molecule as defined herein and at least one (bifunctional) lipidcovalently linked (without a linker) with that RNA molecule. Accordingto a third alternative, the lipid-modified RNA molecule comprises an RNAmolecule as defined herein, at least one linker covalently linked withthat RNA molecule, and at least one lipid covalently linked with therespective linker, and also at least one (bifunctional) lipid covalentlylinked (without a linker) with that RNA molecule. In this context, it isparticularly preferred that the lipid modification is present at theterminal ends of a linear RNA sequence.

Modification of the 5′-End:

According to another preferred embodiment of the invention, a modifiedRNA molecule as defined herein, can be modified by the addition of aso-called “5′ CAP” structure.

A 5′-cap is an entity, typically a modified nucleotide entity, whichgenerally “caps” the 5′-end of a mature mRNA. A 5′-cap may typically beformed by a modified nucleotide, particularly by a derivative of aguanine nucleotide. Preferably, the 5′-cap is linked to the 5′-terminusvia a 5′-5′-triphosphate linkage. A 5′-cap may be methylated, e.g.m7GpppN, wherein N is the terminal 5′ nucleotide of the nucleic acidcarrying the 5′-cap, typically the 5′-end of an RNA. m7GpppN is the5′-CAP structure which naturally occurs in mRNA transcribed bypolymerase II and is therefore not considered as modification comprisedin a modified RNA in this context.

Accordingly, a modified RNA of the present invention may comprise am7GpppN as 5′-CAP, but additionally the modified RNA comprises at leastone further modification as defined herein.

Further examples of 5′-cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety. These modified 5′-CAP structuresare regarded as at least one modification in this context.

Particularly preferred modified 5′-CAP structures are CAP1 (methylationof the ribose of the adjacent nucleotide of m7G), CAP2 (methylation ofthe ribose of the 2nd nucleotide downstream of the m7G), CAP3(methylation of the ribose of the 3rd nucleotide downstream of the m7G),CAP4 (methylation of the ribose of the 4th nucleotide downstream of them7G), ARCA (anti-reverse CAP analogue, modified ARCA (e.g.phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Sequence Modifications:

In a preferred embodiment, the at least one RNA comprises or consists ofa modified RNA molecule having at least one open reading frame, whichencodes at least one peptide or protein. Preferably, the sequence of theopen reading frame in such an RNA molecule is modified as describedherein. Such an RNA comprising a modified sequence of the open readingframe is also included in the term “modified RNA”.

Modification of the G/C Content:

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding region of an RNA is modified, particularlyincreased, compared to the G/C content of its particular wild typecoding region, i.e. the unmodified coding region. The encoded amino acidsequence of the coding region is preferably not modified compared to thecoded amino acid sequence of the particular wild type coding region. Themodification of the G/C-content of the coding region of the modified RNAas defined herein is based on the fact that the sequence of any mRNAregion to be translated is important for efficient translation of thatmRNA. Thus, the composition and the sequence of various nucleotides areimportant. In particular, mRNA sequences having an increased G(guanosine)/C (cytosine) content are more stable than mRNA sequenceshaving an increased A (adenosine)/U (uracil) content. According to theinvention, the codons of the coding region are therefore varied comparedto its wild type coding region, while retaining the translated aminoacid sequence, such that they include an increased amount of G/Cnucleotides. In respect to the fact that several codons code for one andthe same amino acid (so-called degeneration of the genetic code), themost favourable codons for the stability can be determined (so-calledalternative codon usage). Depending on the amino acid to be encoded bythe coding region of the modified RNA as defined herein, there arevarious possibilities for modification of the RNA sequence, e.g. thecoding region, compared to its wild type coding region. In the case ofamino acids, which are encoded by codons, which contain exclusively G orC nucleotides, no modification of the codon is necessary. Thus, thecodons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly(GGC or GGG) require no modification, since no A or U is present. Incontrast, codons which contain A and/or U nucleotides can be modified bysubstitution of other codons which code for the same amino acids butcontain no A and/or U. Examples of these are: the codons for Pro can bemodified from CCU or CCA to CCC or CCG; the codons for Arg can bemodified from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Alacan be modified from GCU or GCA to GCC or GCG; the codons for Gly can bemodified from GGU or GGA to GGC or GGG. In other cases, although A or Unucleotides cannot be eliminated from the codons, it is however possibleto decrease the A and U content by using codons, which contain a lowercontent of A and/or U nucleotides. Examples of these are: the codons forPhe can be modified from UUU to UUC; the codons for Leu can be modifiedfrom UUA, UUG, CUU or CUA to CUC or CUG; the codons for Ser can bemodified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyrcan be modified from UAU to UAC; the codon for Cys can be modified fromUGU to UGC; the codon for His can be modified from CAU to CAC; the codonfor Gln can be modified from CAA to CAG; the codons for Ile can bemodified from AUU or AUA to AUC; the codons for Thr can be modified fromACU or ACA to ACC or ACG; the codon for Asn can be modified from AAU toAAC; the codon for Lys can be modified from AAA to AAG; the codons forVal can be modified from GUU or GUA to GUC or GUG; the codon for Asp canbe modified from GAU to GAC; the codon for Glu can be modified from GAAto GAG; the stop codon UAA can be modified to UAG or UGA. In the case ofthe codons for Met (AUG) and Trp (UGG), on the other hand, there is nopossibility of sequence modification. The substitutions listed above canbe used either individually or in any possible combination to increasethe G/C content of the coding region of the RNA as defined herein,compared to its particular wild type coding region (i.e. the originalsequence). Thus, for example, all codons for Thr occurring in the wildtype sequence can be modified to ACC (or ACG).

Preferably, the G/C content of the coding region of the RNA as definedherein is increased by at least 7%, more preferably by at least 15%,particularly preferably by at least 20%, compared to the G/C content ofthe wild type coding region. According to a specific embodiment at least5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, evenmore preferably at least 80% and most preferably at least 90%, 95% oreven 100% of the substitutable codons in the coding region encoding atleast one peptide or protein, are substituted, thereby increasing theG/C content of said coding region. In this context, it is particularlypreferable to increase the G/C content of the coding region of themodified RNA as defined herein, to the maximum (i.e. 100% of thesubstitutable codons), compared to the wild type coding region.

Codon Optimization:

According to the invention, a further preferred modification of thecoding region encoding at least one peptide or protein of a modified RNAas defined herein, is based on the finding that the translationefficiency is also determined by a different frequency in the occurrenceof tRNAs in cells. Thus, if so-called “rare codons” are present in thecoding region of the wild type RNA sequence, to an increased extent, themRNA is translated to a significantly poorer degree than in the case,where codons coding for relatively “frequent” tRNAs are present. In thiscontext, the coding region of the at least one RNA is preferablymodified compared to the corresponding wild type coding region such thatat least one codon of the wild type sequence, which codes for a tRNAwhich is relatively rare in the cell, is exchanged for a codon, whichcodes for a tRNA which is relatively frequent in the cell and carriesthe same amino acid as the relatively rare tRNA. By this modification,the coding region of the at least one RNA as defined herein, is modifiedsuch that codons, for which frequently occurring tRNAs are available,are inserted. In other words, according to the invention, by thismodification all codons of the wild type coding region, which code for atRNA, which is relatively rare in the cell, can in each case beexchanged for a codon, which codes for a tRNA which is relativelyfrequent in the cell and which, in each case, carries the same aminoacid as the relatively rare tRNA. Which tRNAs occur relativelyfrequently in the cell and which, in contrast, occur relatively rarelyis known to a person skilled in the art; cf. e.g. Akashi, Curr. Opin.Genet. Dev. 2001, 11(6): 660-666. The codons which use for theparticular amino acid the tRNA which occurs the most frequently, e.g.the Gly codon, which uses the tRNA which occurs the most frequently inthe (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content, which is increased, in particular maximized, inthe coding region of the modified RNA as defined herein, with the“frequent” codons without modifying the amino acid sequence of thepeptide or protein encoded by the coding region of the RNA sequence.This preferred embodiment allows provision of a particularly efficientlytranslated and stabilized (modified) RNA sequence as defined herein.

In the context of the present invention, the at least one RNA may alsocomprise a 5′- and/or 3′ untranslated region (5′-UTR or 3′-UTR,respectively). More preferably, the at least one RNA comprises a 5′-CAPstructure as defined above.

Preferably, the at least one RNA further comprises a poly(A) sequence.The length of the poly(A) sequence may vary. For example, the poly(A)sequence may have a length of about 20 adenine nucleotides up to about300 adenine nucleotides, preferably of about 40 to about 200 adeninenucleotides, more preferably from about 50 to about 100 adeninenucleotides, such as about 60, 70, 80, 90 or 100 adenine nucleotides.Most preferably, the at least one RNA comprises a poly(A) sequence ofabout 60 to about 70 nucleotides, most preferably 64 adeninenucleotides.

Preferably, the poly(A) sequence in the at least one RNA is derived froma DNA template by in vitro transcription. Alternatively, the poly(A)sequence may also be obtained in vitro by common methods ofchemical-synthesis without being necessarily transcribed from aDNA-progenitor or may be obtained by enzymatic polyadenylation using apoly(A) polymerase e.g. E. coli poly(A) polymerase.

In addition or as an alternative to a poly(A) sequence as describedabove, the at least one RNA may also comprise a poly(C) sequence,preferably in the region 3′ of the coding region of the RNA. A poly(C)sequence is typically a stretch of multiple cytosine nucleotides,typically about 10 to about 200 cytidine nucleotides, preferably about10 to about 100 cytidine nucleotides, more preferably about 10 to about70 cytidine nucleotides or even more preferably about 20 to about 50 oreven about 20 to about 30 cytidine nucleotides. A poly(C) sequence maypreferably be located 3′ of the coding region comprised by a nucleicacid. In a preferred embodiment of the present invention, the at leastone RNA comprises a poly(A) sequence and a poly(C) sequence, wherein thepoly(C) sequence is located 3′ of the poly(A) sequence.

In a particularly preferred embodiment, the at least one RNA in thecontext of the present invention comprises in 5′-to-3′-direction,optionally a 5′-UTR, an open reading frame, preferably a modified openreading frame as defined herein, a 3′-UTR element, a poly(A) and/or apoly(C) sequence and optionally a histone stem-loop. Preferred 5′-UTR'sare described in WO 2013/143699 and WO 2013/143700, the disclosure ofwhich is herewith incorporated by reference. 3′-UTR sequences, which arepreferred in this context, are described in WO 2013/143698. Examples ofpreferred histone stem-loop sequences are described in WO 2012/019780,whose disclosure is incorporated herewith by reference.

Preferably, the at least one RNA as used herein comprises more than 30nucleotides. More preferably, the at least one RNA is not selected fromsiRNA, small hairpin RNA, microRNA or small nuclear RNA (snRNA). In aparticularly preferred embodiment, the at least one RNA as used hereinis not an siRNA.

Preferably, the at least one RNA is a long-chain RNA. The term‘long-chain RNA’ as used herein herein typically refers to an RNAmolecule, preferably as described herein, which preferably comprises atleast 30 nucleotides. Alternatively, a long-chain RNA may comprise atleast 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,250, 300, 350, 400, 450 or at least 500 nucleotides. A long-chain RNAmolecule may further comprise at least 100 nucleotides, even morepreferably at least 200 nucleotides. A long-chain RNA, in the context ofthe present invention, further preferably comprises from 30 to 50.000nucleotides, from 30 to 20.000 nucleotides, from 100 to 20.000nucleotides, from 200 to 20.000 nucleotides, from 200 to 15.000nucleotides or from 500 to 20.000 nucleotides. The term long-chain RNA'as used herein is not limited to a certain type of RNA, but merelyrefers to the number of nucleotides comprised in said RNA. In apreferred embodiment, the at least one RNA as used herein is along-chain mRNA.

According to the invention, the method comprises a step a), whichcomprises providing a first liquid composition comprising at least oneRNA.

In this context, the first liquid composition may comprise exactly one(type of) RNA molecule, or a mixture of two or more different (types of)RNA molecules, such as, for example, two, three, four, five, six etc.different (types of) RNA molecules, wherein a plurality of each (typeof) RNA molecule is preferably present in the first liquid composition.Preferably, the first liquid composition comprises from 1 to 20different RNA molecules, further preferred from 1 to 10 different RNAmolecules, further preferred from 1 to 6, and still further preferred 1,2, 3, 4, 5 or 6 different RNA molecules. In a preferred embodiment, thefirst liquid solution comprises more than one RNA, wherein the RNAmolecules differ in their respective coding regions and, optionally,further structural elements. Preferably, the first liquid compositioncomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different RNA molecules, eachof which encodes a distinct peptide or protein preferably an antigen.Especially preferably, the first liquid composition comprises one RNA.

Preferably, the first liquid composition comprises water as a solvent.Additionally, the first liquid composition may comprise at least onesolvent miscible with water. Examples of such water-miscible solventsare known in the art. Preferred examples of additional solvents arealcohols, such as, e.g. ethanol, etc., DMSO, and the like. Especiallypreferred, the additional solvent is pharmaceutically acceptable.Examples of pharmaceutically acceptable solvents are, e.g. ethanol, etc.

Preferably, the first liquid composition is an aqueous solution of RNAand, optionally, at least one further component. In a preferredembodiment, the at least one RNA is present in the first liquidcomposition in its free form, i.e. as ‘naked’ RNA.

Preferably, the first liquid composition comprises RNA in aconcentration of from 0.1 to 20 g/L, preferably from 0.5 to 10 g/L, morepreferably from 0.5 to 7 g/L, more preferably of from 0.5 to 2 g/L andmost preferably of from 0.5 to 1.0 g/L. More preferably, the firstliquid composition comprises the at least one RNA as defined herein in aconcentration as defined above.

The inventive method further comprises a step b) of providing a secondliquid composition comprising at least one cationic or polycationiccompound.

In the context of the invention, the term ‘cationic or polycationiccompound’ is used for a compound, preferably an oligomeric or polymericcompound, comprising one to numerous cationic functions (i.e. positivecharges). Such compounds are known in the art, where they are sometimesalso referred to as “polycationic molecules” or “polycationic polymers”.Examples of cationic or polycationic compounds comprise cationic orpolycationic peptides or polypeptides, cationic or polycationicproteins, cationic or polycationic polyamino acids, cationic orpolycationic carbohydrates, cationic or polycationic synthetic polymers,cationic or polycationic small synthetic organic molecules, inorganicmultivalent cations, cationic lipids, and the like.

Preferred examples of cationic or polycationic compounds which can beused in the method of the present invention are disclosed, e.g. in WO2008/077592 A1, WO 2009/095226 A2, WO 2010/037539 and WO 2011/026641 A1,which are all incorporated herein by reference.

The second liquid composition provided in step b) comprises at least onecationic or polycationic compound, wherein the at least one cationic orpolycationic compound is preferably capable of forming a complex withthe at least one RNA comprised in the first liquid composition providedin step a) of the inventive method. More preferably, the at least onecationic or polycationic compound comprised in the second liquidcomposition forms a nanoparticle with the at least one RNA comprised inthe first liquid composition, wherein the composition, optionallycomprises at least one further component, such as, for instance, alyoprotectant, preferably as defined herein.

Alternatively or in addition to a cationic or polycationic compound, theat least one RNA may also be complexed by a compound selected from thegroup of polymers or complexing agents, typically comprising, withoutbeing limited thereto, any polymer suitable for the preparation of apharmaceutical composition, such as minor/major groove binders, nucleicacid binding proteins, lipoplexes, nanoplexes, non-cationic ornon-polycationic compounds, such as PLGA, Polyacetate, Polyacrylate,PVA, Dextran, hydroxymethylcellulose, starch, MMP, PVP, heparin, pectin,hyaluronic acid, and derivatives thereof. In a further preferredembodiment, the at least one RNA may also be complexed—alternatively orin addition to a cationic or polycationic compound—by a compoundselected from the group of polymers or complexing agents, typicallycomprising, without being limited thereto, any polymer suitable for thepreparation of a pharmaceutical composition, such as minor/major groovebinders, nucleic acid binding proteins, lipids, lipoplexes, nanoplexes,non-cationic or non-polycationic compounds, such as PLGA, Polyacetate,Polyacrylate, PVA, Dextran, hydroxymethylcellulose, starch, MMP, PVP,heparin, pectin, hyaluronic acid, and derivatives thereof.

Preferably, the second liquid composition comprises water as a solvent.Additionally, the second liquid composition may comprise one or more ofsolvent miscible with water. Examples of such water-miscible solventsare known in the art. Preferred examples of additional solvents arealcohols, such as, e.g. ethanol, etc., DMSO, and the like. Especiallypreferred, the additional solvent is pharmaceutically acceptable.Examples of pharmaceutically acceptable solvents are, e.g. ethanol, etc.

Preferably, the second liquid composition is an aequeous solution of theat least one cationic or polycationic compound, which optionallycomprises at least one further component, wherein the at least onefurther compound is preferably a lyoprotectant as defined herein.

Preferably, the second liquid composition comprises a cationic orpolycationic compound in a concentration of from 0.05 to 10.00 g/L,preferably of from 0.10 to 5.00 g/L, more preferably of from 0.10 to 1.0g/L and most preferably of from 0.1 to 0.5 g/L.

In a preferred example, the second liquid composition comprisesprotamine in a concentration of from 0.1 to 1 g/L, more preferably in aconcentration of 0.3 to 0.6 g/L or in a concentration of from 0.4 to 0.5g/L and most preferably in a concentration of from 0.4 to 0.5 g/L andeven more preferably in a concentration of from 0.3 to 0.45 g/L.

Preferably, the concentration of cationic or polycationic compound inthe second liquid composition is adjusted to provide a predeterminedratio with respect to the concentration of RNA in the first liquidcomposition.

Preferably, the concentration of cationic or polycationic compound isadjusted to the concentration of the RNA so as to provide an N/P-ratioof about 0.1-10, preferably in a range of about 0.3-4 and mostpreferably in a range of about 0.5-2 or 0.7-2 regarding the ratio ofmRNA:cationic or polycationic compound and/or polymeric carrier,preferably as defined herein, in the complex, and most preferably in arange of about 0.7-1.5, 0.5-1 or 0.7-1, and even most preferably in arange of about 0.3-0.9 or 0.5-0.9. In this context, the N/P-ratio isdefined as the ratio of the number of cationic nitrogen functions of thecationic or polycationic compound (N) to the number of phosphateresidues of the RNA (P). It may be calculated on the basis that, forexample, 1 μg RNA typically contains about 3 nmol phosphate residues,provided that the RNA exhibits a statistical distribution of bases.

The concentration of a cationic or polycationic compound, preferablyprotamine, in the second liquid composition is preferably such that theweight ratio of the cationic or polycationic compound, preferablyprotamine, and the at least one RNA are present in the mixture of thefirst and the second liquid composition, which is obtained in step c) ofthe inventive method, at a weight ratio (RNA:cationic or polycationiccompound, w/w) in a range from 6:1 (w/w) to about 0.25:1 (w/w), morepreferably from about 5:1 (w/w) to about 0.5:1 (w/w), even morepreferably of about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1 (w/w)to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) toabout 2:1 (w/w). Most preferably, the weight ratio of the at least oneRNA to the at least one cationic or polycationic compound, preferablyprotamine, in the mixture obtained in step c) is 2:1 (w/w).

According to the invention, the at least one cationic or polycationiccompound in the second liquid composition may be a cationic orpolycationic peptide or protein, which optionally comprises or isadditionally modified to comprise at least one —SH moiety. Preferably,the at least one cationic or polycationic compound is selected fromcationic peptides having the following sum formula (V):{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (SEQ ID NO:2);  formula (V)

wherein l+m+n+o+x=3-100, and l, m, n or o independently of each other isany number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90 and 91-100 provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide; and Xaa is any amino acidselected from native (=naturally occurring) or non-native amino acidsexcept of Arg, Lys, His or Orn; and x is any number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide. Any of amino acids Arg, Lys, His, Orn and Xaa may bepositioned at any place of the peptide. In this context, cationicpeptides or proteins in the range of 7-30 amino acids are particularpreferred.

Further, the cationic or polycationic peptide or protein, when definedaccording to formula {(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}(formula (V)) as shown above and which comprises or is additionallymodified to comprise at least one —SH moeity, may be, without beingrestricted thereto, selected from subformula (Va):{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)(Cys)_(y)} (SEQ ID NO:3)  subformula (Va)

wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys, His, Orn or Cysand y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,71-80 and 81-90, provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide. Further, the cationic orpolycationic peptide may be selected from subformula (Vb):Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys2 (SEQ ID NO:4)  subformula (Vb)

wherein empirical formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (V)) is asdefined herein and wherein Cys, and Cys2 are Cysteines proximal to, orterminal to (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x).

In the context of the inventive method, particularly preferred cationicor polycationic compounds include protamine, nucleoline, spermine orspermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, oligoarginines as defined herein,such as Arg₇, Arg₈, Arg₉, Arg₇, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, CR₁₂C,CR₁₂, (RKH)₄, Y(RKH)₂R, etc., basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs),PpT620, proline-rich peptides, arginine-rich peptides, lysine-richpeptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histones.In a particularly preferred embodiment, the cationic or polycationiccompound is protamine.

Further preferred cationic or polycationic compounds, which may becomprised in the second liquid composition according to the inventivemethod, may include cationic polysaccharides, for example chitosan,polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationiclipids, e.g. DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammoniumchloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP,DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB,DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI:Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)-propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g polyethyleneglycole); etc.

Preferably, the first liquid composition and/or the second liquidcomposition further comprise at least one compound selected from a saltor a lyoprotectant.

Preferably, the first liquid composition and/or the second liquidcomposition comprise at least one salt selected from the groupconsisting of NaCl, KCl, LiCl, MgCl₂, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄,Na₃PO₄, KI, KBr, K₂CO₃, KHCO₃, K₃PO₄, K₂SO₄, CaCl₂, CaI₂, CaBr₂, CaCO₃,CaSO₄, Ca(OH)₂, and Ca₃(PO₄)₂.

Preferably, the first liquid composition and/or second liquidcomposition comprise at least one cation, preferably at least one cationselected from the group consisting of Na³⁰, K⁺, Li⁺, Mg²⁺, Ca²⁺ andBa²⁺. Preferred examples of cations and corresponding salts are Na⁺(e.g. NaCl), K⁺ (e.g. KCl), Li⁺ (LiCl) or Mg²⁺ (MgCl₂), particularlypreferred is Na⁺.

The first liquid composition and/or second liquid composition maycomprise at least one cation in a concentration of up to 50 mM,preferably of from 0.001 to 50 mM, more preferably from 3 to 30 mM, morepreferably from 3 to 20 mM, further preferably from 5 to 15 mM, and mostpreferred from 5 to 10 mM. It is particularly preferred to add cationsin a concentration of at least 3 mM to the first liquid composition.Preferably, the content of cations in the first and/or second liquidcomposition is 30 mM or less, more preferably from 3 to 30 mM, morepreferably from 4 to 26 mM, and especially preferred from 5 to 10 mM.Preferably, the first and/or second liquid composition may comprise Na⁺cations in a content of from 4 to 26 mM, more preferably from 5 to 10mM. In a preferred example, the Na⁺ content is 9 mM.

In a particularly preferred embodiment, the first liquid compositionand/or the second liquid composition comprises a cation in aconcentration from 0.1 to 10 mM, more preferably from 1 to 5 mM. In apreferred example, the cation concentration in the first liquidcomposition and/or the second liquid composition is 4.6 mM.

More preferably, the first liquid composition and/or the second liquidcomposition comprises an anion in a concentration from 0.1 to 10 mM,more preferably from 1 to 5 mM. In a preferred example, the anionconcentration in the first liquid composition and/or the second liquidcomposition is 4.6 mM.

Preferably, the ratio of cation to RNA in the first liquid compositionis from 3 to 30 mmol cation/g RNA, preferably from 4 to 23 mmol cation/gRNA, and most preferably from 5 to 15 mmol cation/g RNA. It isparticularly preferred to add cations in a ratio of a least 3 mmolcations/g RNA. Preferably, the first liquid composition comprises Na⁺cations and RNA in a ratio of from 4.6 to 27.5 mmol Na⁺/g RNA, morepreferably from 5 to 15 mmol Na⁺/g RNA and most preferably from 6 to 11mmol Na⁺/g RNA. In a preferred example, the first liquid compositioncomprises Na⁺ cations and RNA in a ratio of 10.3 mmol Na⁺/g RNA.

In another preferred embodiment, the first liquid composition comprisingRNA does not comprise additional cations.

In a particularly preferred embodiment, the second liquid compositioncomprises Na⁺ cations in a concentration from 0.1 to 10 mM, morepreferably from 1 to 5 mM. In a preferred example, the Na⁺ concentrationof the second liquid composition is 4.6 mM.

Preferably, the first liquid composition and/or second liquidcomposition comprise at least one anion selected from the groupconsisting of Cl⁻, CO₃ ²⁻, PO₄ ³⁻ and SO₄ ²⁻. Preferably, theconcentration of the anion in the first liquid composition and/or secondliquid composition is 23 mM or less, more preferably from 1.5 to 23.0mM, and especially preferred from 5.0 to 10.0 mM. Preferably, the firstliquid composition and/or the second liquid composition may comprise Cl⁻anions in a content of from 1.5 to 23 mM, more preferably from 5.0 to10.0 mM. In a preferred example, the Cl⁻ content is 6.5 mM.

In a preferred embodiment, the first liquid composition comprising RNAdoes not comprise additional anions.

In another particularly preferred embodiment, the second liquidcomposition comprises Cl⁻ anions in a concentration from 0.1 to 10 mM,more preferably from 1 to 5 mM. In a preferred example, the Cl⁻concentration of the second liquid composition is 4.6 mM.

Preferably, the first liquid composition and/or second liquidcomposition comprise at least one component selected from acryoprotectant, a lyoprotectant or a bulking agent. In this context,cryoprotectants are understood as excipients, which allow influencingthe structure of a frozen material and/or the eutectical temperature ofthe mixture. Lyoprotectants are typically excipients, which partially ortotally replace the hydration sphere around a molecule and thus preventcatalytic and hydrolytic processes. A bulking agent (e.g. a filler) isany excipient compatible with the RNA and/or the cationic orpolycationic compound. As used herein, a bulking agent may be used forincreasing the volume and/or the mass of the liquid compositions. Inaddition, a bulking agent may also protect the at least one RNA fromdegradation.

Preferably, the first liquid composition and/or the second liquidcomposition comprise at least one lyoprotectant. In a particularlypreferred embodiment, the second liquid composition comprises at leastone lyoprotectant, preferably as defined herein. According to oneembodiment, the lyoprotectant is selected from the group consisting ofsucrose, mannose, trehalose, mannitol, polyvinylpyrrolidone, and Ficoll70. Alternatively, the lyoprotectant may be selected from the groupconsisting of glucose, fructose, sucrose, mannose, trehalose, mannitol,polyvinylpyrrolidone, and Ficoll 70.

Preferably, the concentration of lyoprotectant in the first liquidcomposition and/or the second liquid composition is in a range of about0.01 to about 40% (w/w), preferably of about 0.01 to about 30% (w/w),more preferably of about 0.1 to about 20% (w/w), even more preferably ofabout 0.5 to about 20% (w/w), and most preferably of about 2.5 to about20% (w/w), e.g. of about 5 to about 20% (w/w), such as about 5% or 10%(w/w). More preferably, the concentration of a lyoprotectant, preferablyas defined herein, in the second liquid composition is in a range ofabout 0.01 to about 40% (w/w), preferably of about 0.01 to about 30%(w/w), more preferably of about 0.1 to about 20% (w/w), even morepreferably of about 1 to about 20% (w/w), and most preferably of about 5to about 15% (w/w), e.g. of about 8 to about 14% (w/w), such as about10% (w/w)

Preferably, the first liquid composition and/or the second liquidcomposition comprise at least one component selected from the group of(free) carbohydrates. Such group of (free) carbohydrates may comprise,without being limited thereto, any (free) carbohydrate, suitable for thepreparation of a pharmaceutical composition, preferably, without beinglimited thereto, (free) monosaccharides, such as e.g. (free) glucose,(free) fructose, (free) galactose, (free) sorbose, (free) mannose(“free” preferably means unbound or unconjugated, e.g. the mannose isnot covalently bound to the at least one RNA, or in other words, themannose is unconjugated, preferably with respect to the at least oneRNA), etc., and mixtures thereof; disaccharides, such as e.g. lactose,maltose, sucrose, trehalose, cellobiose, etc., and mixtures thereof;polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,starches, etc., and mixtures thereof; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol, pyranosyl sorbitol,myoinositol, etc., and mixtures thereof. Examples of sugars that arepreferably used in the composition according to the invention includelactose, sucrose or trehalose. Generally, a sugar that is preferred inthis context, has a high water displacement activity and a high glasstransition temperature. Furthermore, a sugar suitable for use in thefirst and/or second liquid composition is preferably hydrophilic but nothygroscopic. In addition, the sugar preferably has a low tendency tocrystallize, such as trehalose. Trehalose is particularly preferred.

According to a preferred embodiment, the first liquid composition and/orthe second liquid composition comprise a carbohydrate component,preferably a sugar, more preferably trehalose. In a preferredembodiment, a carbohydrate component, preferably a sugar, morepreferably trehalose is present in the liquid provided in step a) of themethod at a concentration of about 0.01 to about 40% (w/w), preferablyin a concentration of about 0.01 to about 30% (w/w), more preferably ina concentration of about 0.1 to about 20% (w/w), even more preferably ina concentration of about 0.5 to about 20% (w/w), and most preferably ina concentration of about 2.5 to about 20% (w/w), e.g. at a concentrationof about 5 to about 20% (w/w), such as about 10% or 5% (w/w). In aparticularly preferred embodiment, the second liquid compositioncomprises a carbohydrate component, preferably a sugar, more preferablytrehalose. In a preferred embodiment, a carbohydrate component,preferably a sugar, more preferably trehalose is present in the liquidprovided in step a) of the method at a concentration of about 0.01 toabout 40% (w/w), preferably in a concentration of about 0.01 to about30% (w/w), more preferably in a concentration of about 0.1 to about 20%(w/w), even more preferably in a concentration of about 1 to about 20%(w/w), and most preferably in a concentration of about 5 to about 15%(w/w), e.g. at a concentration of about 8 to about 14% (w/w), such asabout 10% (w/w)

Preferably, the first liquid composition is an aqueous solution,preferably having a pH of from 4 to 9, more preferably from 5 to 7. In apreferred example, the pH of the first liquid composition is 5.8.

Preferably, the second liquid composition is an aqueous solution,preferably having a pH of from 4 to 9, more preferably from 6 to 8. In apreferred example, the pH of the second liquid composition is about 7.

Preferably, the first liquid composition and the second liquidcomposition are provided in a reservoir vessel each, which optionallymay be held at a predetermined temperature and/or pressure usingappropriate temperature-controlling and pressure-controlling means,respectively, known in the art.

The respective reservoir vessel can be connected to the reactor via anyconnection means known in the art, e.g. by means of a tube, a hose,tubing, or a pipe.

Materials suitable for the reservoir vessels and connection means areknown in the art, and may be the same as those used for the reactor ormixing means as described herein.

According to step c) of the method of the present invention, the firstliquid composition comprising at least one RNA and the second liquidcomposition comprising at least one cationic or polycationic compoundare introduced into at least one reactor, wherein the first liquidcomposition comprising at least one RNA and the second liquidcomposition comprising at least one cationic or polycationic compoundare mixed with a blend time of 5 seconds or less. Preferably, the firstliquid composition and the second liquid composition are introduced intoat least one reactor in a controlled manner.

Step c) preferably comprises reacting the at least one RNA with the atleast one cationic compound. In this context, the term ‘reacting’typically relates to the formation of a complex, preferably ananoparticle, by the at least one RNA and the at least one cationic orpolycationic compound.

The first liquid composition and the second liquid composition can beintroduced into the reactor by any method known in the art. For example,the first liquid composition and the second liquid composition can beintroduced into the reactor by pumping or gravity flow. Preferably, thefirst liquid composition and the second liquid composition areintroduced into the reactor by pumping at a controlled flow rate.

Pumping of the first liquid composition and the second liquidcomposition can be carried out using any pump known in the art, forexample, a syringe pump, such as TSE systems 540060-B, NE-1000 SingleSyringe Pump from New Era Pump Systems, Inc.or a KNAUER Smartline Pump1000 or a peristaltic pump, such as a Watson Marlow Pump 323S or amembrane pump/diaphragm pump such as a Lever Pump or a quattroflow pump.

The first liquid composition and/or the second liquid composition may beintroduced into the reactor at any suitable flow rate. Optimum flowrates may be readily determined by a skilled person for any reactorsystem used.

Preferable flow rates are 0.1 ml/minute or more, more preferably 0.3ml/minute or more, more preferably 0.5 ml/minute or more, preferably 1.0ml/minute or more, more preferably 1.5 ml/minute or more, or morepreferably 10 ml/minute or more, 50 ml/minute or more, 100 ml/minute ormore, or 500 ml/minute or more, and most preferably 750 ml/minute ormore. In a preferred embodiment, the flow rate is in a range from about0.1 ml/minute to 1500 ml/minute, preferably from 0.3 ml/minute to 1000ml/minute, from 5 ml/minute to 700 ml/minute or from 5 ml/minute to 500ml/minute. Further preferred flow rates are 5 ml/minute or more, morepreferably 10 ml/minute or more, more preferably 50 ml/minute or more,and more preferably 250 ml/minute or 500 ml/minute or more, and mostpreferably 750 ml/minute or more. More preferably, flow rates are in therange from about 50 ml/minute to 200 ml/minute, even more preferably inthe range from about 100 ml/minute to 200 ml/minute. Most preferably theflow rate is about 160 ml/minute.

In a preferred embodiment, the first liquid composition and/or thesecond liquid composition are introduced into the at least one reactorwith a flow rate of 0.1 ml/minute or more, 1 ml/minute or more, 10ml/minute or more, preferably 50 ml/minute or more, more preferably 100ml/minute or more, more preferably 150 ml/minute or more, morepreferably 300 ml/minute or more, more preferably 800 ml or more, mostpreferably in a range of 100-800 ml/minute via a pump device.

Preferably, the first liquid composition and/or the second liquidcomposition are introduced into the reactor at the same flow rate.

In a particularly preferred embodiment, the first liquid composition andthe second liquid composition are concurrently introduced into the atleast one reactor, preferably under controlled conditions e.g. atconstant flow rates.

In the context of the invention, the term “controlled conditions” or“introduced into the at least one reactor, preferably under controlledconditions” typically refers to stable, non-variable conditions that areapplied to introduce the first liquid composition and the second liquidcomposition into the reactor, e.g. a mixer as described herein. These“controlled conditions” may be controlled flow rate, liquid flow, pumprate, pump flow, injection rate, injection flow, pressure, temperatureand the like. Preferably, the term “controlled conditions” relates toconstant flow rates and/or constant liquid flow.

Preferably, the first liquid composition and the second liquidcomposition are introduced into the at least one reactor so that the atleast one RNA and the at least one cationic or polycationic compound arepresent in the reactor with a N/P-ratio (defined as above) of about0.1-10, preferably in a range of about 0.3-4 and most preferably in arange of about 0.5-2 or 0.7-2 regarding the ratio of mRNA:cationic orpolycationic compound and/or polymeric carrier, preferably as definedherein, in the complex, and most preferably in a range of about 0.7-1.5,0.5-1 or 0.7-1, and even most preferably in a range of about 0.3-0.9 or0.5-0.9.

In the context of the invention, the term ‘reactor’ is used for a vesselproviding a reaction space or volume (sometimes also referred to as‘reaction chamber’), wherein the first liquid composition comprising atleast one RNA and the second liquid composition comprising at least onecationic or polycationic compound are mixed and reacted. The reactor mayby an open vessel or a closed vessel, without limitation regarding theshape or the dimension of the vessel.

In the context of the present invention, the reactor may optionallycomprise appropriate temperature-controlling and/or pressure-controllingmeans, respectively, to control a predetermined temperature and/orpressure within the reactor. Suitable temperature-controlling andpressure-controlling means are known in the art. The reaction space istypically defined by the walls of the reactor and the fill level. If areactor is operated in a completely filled state, the reaction space(reaction chamber) corresponds to the total liquid volume held by thereactor, wherein the total liquid volume is preferably defined by theenclosing walls of the reactor.

Preferably, an open vessel comprises an opening at its upper side,preferably an open top. The open reactor can be advantageously filledand emptied through its opening. The reactor may comprise inlet and/oroutlet ports. If the reactor is a closed vessel, the reactor willcomprise appropriate inlet and outlet ports for introducing the firstand second liquid compositions, and for recovering the product liquidcomposition, respectively. Such ports are preferably designed to controlthe inlet or outlet flow, respectively. Such ports are known in the artand may be of any kind suitable for the purpose of controlling the flowof a liquid composition. Optionally, the reactor may also compriseadditional components, such as (safety) valves, etc.

The method of the present invention is preferably conducted at atemperature in a range of from 4 to 50° C., more preferably from 10 to30° C., and most preferably at room temperature, about 20° C.Accordingly, the reactor system preferably comprises appropriatetemperature-controlling means (e.g. cooling and/or heating means,temperature sensors, and the like), which allow to conduct the method ofthe invention in a temperature range of from 4 to 50° C., morepreferably from 10 to 30° C. and most preferably at about 20° C. (roomtemperature). Preferably, the first liquid composition, the secondliquid composition and/or the mixture of the two in the reaction chamberhave a temperature as defined above. Suitable means for cooling and/orheating such a system or parts of such a system are known in the art.

The reactor and its components may be manufactured from any suitablematerial, preferably from a material that is inert to any reagent,solvent and product of the reaction to be carried out within thereactor. For example, the reactor material should be inert to both acidsand bases as well as to any suitable solvent. Suitable materials for thereactor include, for instance, glass, metals, such as, e.g., steel,preferably stainless steel, polymeric compounds, such as, e.g.,polyolefins, for example, polyethylene, fluorine-containing polymericcompounds, for example, polytetrafluoroethylene (PTFE, Teflon®),polypropylene, polyvinylidene fluoride (PVDF), polyvinyl chloride (PVC),chlorinated polyvinyl chloride (CPVC), and polyacetal (polyoxymethylene)Preferred materials are polymeric compounds, glass or steel.

Preferably, the at least one reactor is one single reactor.

Preferably, the reactor has a volume in a range from 0.1 ml to 10 l,preferably from 0.5 ml to 5 l, more preferably from 1 ml to 500 ml andmost preferably from 1 ml to 100 ml.

The reactor may be of any type and design known in the art, as long asthe reactor is provided with a design allowing the mixing and reactingof the first and second liquid compositions under controlled conditions,preferably the simultaneous mixing and reacting of the first and secondliquid compositions under controlled conditions. The term ‘simultaneousmixing and reacting’ means that the first and second liquid compositionsare thoroughly mixed and, at the same time, the reaction between the atleast one RNA in the first liquid composition and the at least onecationic or polycationic compound in the second liquid compositions isconducted, i.e. started and completed. In other words, the reactordesign provides a reaction space (reaction chamber), wherein thereaction between the at least one RNA and the at least one cationic orpolycationic compound is completed under thoroughly controlledconditions.

According to an embodiment of the present invention, the reactor is atank reactor. As used herein, the term ‘tank reactor’ is typically usedfor a reactor, which comprises an open or closed vessel, such as, forinstance a container of any shape or size, in which the first liquidcomposition and the second liquid composition are mixed and reacted asdescribed herein.

The first liquid composition and/or second liquid composition may beintroduced into the reactor and/or recovered from the reactor as a bulk,preferably in a discontinuous process. Alternatively, the first and/orsecond liquid composition may be introduced into the reactor and/orrecovered from the reactor by a suitable inlet and/or outlet,respectively, preferably in a continuous process. In specificembodiments, the first and/or second liquid composition preferably flowthrough the reactor (such as, for example, a tube reactor), wherein theliquid compositions are mixed and reacted as described herein.

According to step d) of the inventive method, the product liquidcomposition comprising the nanoparticle comprising the at least one RNAand the at least one cationic or polycationic compound is recovered fromthe reactor. In a preferred embodiment, the product liquid compositionis recovered after completion of the reaction. Thereby, the methodaccording to the present invention produces a nanoparticle, which ischaracterized by uniform average particles size and polydispersity,reliably, under controlled conditions.

By the method according to the present invention, the formation ofunwanted side products resulting from unwanted side reactions is reducedor altogether avoided. It was found that a product liquid composition,for which the presence of unwanted side products resulting from unwantedside reactions can be detected by measurement of absorption and/orturbidity directly after the production thereof, is of poor quality withrespect to the desired nanoparticle or is likely to deterioratesubsequently, for instance by formation of aggregates or precipitates.In particular, it was found that such unwanted side products and/or sidereactions adversely affect the stability of the product liquidcomposition, especially upon (long term) storage, which is a productproperty required for pharmaceutical applications. An uncontrollableformation of precipitates would lead to compositions havinguncontrollable concentrations of the active ingredient (RNA-comprisingcompound and/or nanoparticle), which is completely unacceptable forpharmaceutical applications. However, the product liquid compositionsobtained according to the method of the present invention were found tobe of superior quality, stability and unaffected from such unwanted sideproducts and/or side reactions.

It was found that the presence of unwanted side products (e.g.precipitates) resulting from unwanted side reactions results in areduction of clarity, preferably as determined by absorptionmeasurement, preferably at a wavelength of 350 nm, and/or an increase ofturbidity of the product composition, so that the measurement ofabsorption and/or turbidity of the product liquid composition provides afast and reliable way for detecting unwanted side products resultingfrom unwanted side reactions. A measurement of absorption and/orturbidity can easily be integrated into a production process, even whenoperated continuously. A person skilled in the art will readily know howto incorporate the appropriate measurements of absorption and/orturbidity into the reactor system and/or production method according tothe present invention.

Thus, the method according to the present invention produces ananoparticle, which is characterised by uniform average particles sizeand polydispersity, reliably under controlled conditions, withoutallowing unwanted side reactions resulting in unwanted side products andstability issues caused thereby, independent of the scale of production.Further, the method according to the present invention is bothcost-effective and reliable, particularly on a large scale, whichrenders the method of the invention especially suitable for thepharmaceutical production of RNA-comprising nanoparticles at anindustrial scale.

According to the invention, the at least one reactor is characterised bya blend time of 5 seconds or less. Preferably, the blend time is 2.5seconds or less, more preferably 2.0 seconds or less, more preferably,1.0 second or less, more preferably 0.5 second or less, more preferably0.25 second or less, more preferably 0.1 seconds or less and mostpreferably 0.05 seconds or less. Alternatively, the blend time ispreferably in a range from about 0.001 seconds to about 5 seconds, morepreferably from about 0.01 seconds to about 5 seconds, even morepreferably from about 0.1 seconds to about 5 seconds and most preferablyfrom about 0.001 to about 2 seconds or from about 0.01 to about 2seconds.

The term “blend time” (also referred to as “mixing time” or“macro-mixing time” in the art) is commonly used in the art to indicatethe time required to reach a predefined degree of homogeneity in avessel or reactor (also referred to as ‘uniformity’) under predeterminedreaction conditions. The blend time is known to depend on the reactordesign as well as various operational conditions of the reactor, forexample, on size and geometry of the reaction chamber, size and geometryof an optional mixer and/or stirrer, the mixing or stir rate, size andgeometry of optional baffles, energy input, the flow rate of theindividual solutions, viscosity of the solutions, temperature, and thelike, which are readily adjusted accordingly by the skilled person.

In the context of the present invention, “blend time” typically refersto the time required reaching homogeneity (uniformity) of the mixture ofat least 50%. Preferably, the homogeneity (uniformity) is 50% or more,more preferably 60% or more, more preferably 70% or more, morepreferably 80% or more and most preferable 90% or more.

Methods for the experimental determination and semi-theoreticalprediction of the blend time in reactors (such as mixers) are known inthe art. Typically, these methods consist of adding a small amount of amiscible tracer to the liquid in the reactor (e.g. a mixer), andmonitoring the concentration of the tracer with time at differentlocations throughout the system. Examples of tracers comprise tracersthat can be detected by optical means (e.g. by color and/or absorptionmeasurements, or the like), by conductivity measurements (e.g. bymeasurement of the electrical or thermal conductivity, or the like), byradioactivity, and the like. Typically, the tracer concentration at agiven point in the system fluctuates with time. However, the amplitudesof the concentration fluctuations decrease over time, and eventually thelocal concentration converges asymptotically toward the ultimatehomogeneous concentration value corresponding to a uniform dispersion ofthe tracer in the entire system. Preferably, when using a colorimetrictracer, image processing of digitized images of the mixing system can beused, in combination with imaging processing software, to detect a colorchange at a particular location in the vessel very precisely. The blendtime is defined as the time required by the tracer liquid system toreach a desired and predefined level of tracer concentration uniformity(homogeneity) at a predefined location. Usually, a 70%, 80%, 85%, 90% or95% uniformity level (homogeneity) is defined as the endpoint of theblending process.

Preferably, the blend time is experimentally determined, preferably by amethod selected from a colorimetric method, a method based onconductivity measurements, and a chemical method. Alternatively, theblend time may be determined experimentally, preferably by a methodselected from a colorimetric method, a method based on conductivitymeasurements, a chemical method, or by simulation, e.g. by computationalfluid dynamics (CFD).

An example of a colorimetric method for determining the blend timeexperimentally comprises a method, wherein at least one misciblecoloured tracer compound (e.g. a coloring agent, a dye, a pigment, orthe like) is added to the composition in the reactor, preferably beforeor concurrently with the introduction of the first liquid compositionand the second liquid composition, and the change of color is detectedin the whole reactor or a definite zone by means of an optical detector.The blend time is the time interval from the introduction of the tracerto the time, when a predetermined uniformity level (homogeneity) of thedistribution of the tracer is detected (e.g., a 95% uniformity level,usually chosen in the art).

An example for a method based on conductivity measurements fordetermining the blend time experimentally comprises using a measurementof a physical quantity (e.g. measurement of the thermic or electricconductivity, or the like) to detect the concentration and/ordistribution of at least one miscible tracer (e.g., an electrolyte, orthe like). The tracer is added to the composition in the reactor, andthe change of a physical quantity, which is proportinal to theconcentration of the tracer, is measured in the whole reactor or in adefinite zone by means of one or more probes. The blend time is the timeinterval from the introduction of the tracer to the time, when apredetermined uniformity level (homogeneity) of the distribution of thetracer is detected (e.g., a 95% uniformity level, usually chosen in theart).

A method for determining the blend time using a radioactive tracer iscarried out in a manner analogous to the colorimetric and/orconductimetric method, wherein the concentration of the at least oneradioactive tracer is measured in the whole reactor or in definite zonesby means of a suitable detector.

An example for a chemical method for determining the blend timeexperimentally comprises using a chemical reaction, preferably aninstantaneous chemical reaction (e.g. an acid-base-type reaction inaqueous systems), wherein a suitable first reactant (for example, anacid solution, such as, e.g. aqueous HCI, or the like) is added instoichiometric excess to the composition in the reactor comprising asuitable second reactant (for example, a basic solution, such as, e.g.aqueous NaOH, or the like). The blend time is the time necessary toachieve equivalence conditions in the whole reactor or a definite zone,wherein the equivalence point can preferably be determined by means of acolor change of a suitable indicator (colorimetric method), ameasurement of conductivity, a measurement of pH, or the like. Therespective detection can be carried out in the same way as described forthe colorimetric and conductimetric methods, including all detectors,probes, conditions, etc. utilized therein.

A further example of a chemical method for determining the blend timeexperimentally comprises the use of two or more indicators or tracers. Apreferred example for a chemical method using two indicatorssimultaneously is the DISMT method (Dual Indicator System for MixingTime) as described in Melton et al. (DISMT-Determination of mixing timethrough color changes, Chemical Engineering Communications Volume 189,Issue 3, 2002). This method uses a semi-quantitative visualization ofliquid/liquid mixing processes in color changes. In DISMT, two liquids,one red and one blue, are mixed to produce a yellow liquid. Throughappropriate choice of the acid-base indicators used, and the initialpH's of the two solutions, the yellow liquid appears only in thoseregions where the mixing fraction is within a designated fractionaldeviation (say 5%) of the infinite time mixing fraction. Thus, the 95%mixing time/distance can be defined, for the whole volume of the mixingsystem, as the time/distance for all of the liquid to become yellow.DISMT makes use of two standard acid-base indicators, methyl red (red toyellow, pK˜5) and thymol blue (yellow to blue, pK˜9). Both indicatorsare added to both unmixed solutions. A red (acidic) solution is mixedwith a blue (basic) solution, and in those regions where the mixingfraction is within 5% of the mixing fraction at infinite time, thesolution is yellow. With a clear mixing vessel, an observer may seedistinct red and blue regions as well as the later emergence of yellowregions. With DISMT, the 95% mixing time for the entire vessel may bedefined as the time for the entire liquid volume to become yellow. Thefundamental idea underlying the DISMT method is that two color changesare used to mark two acid-base mixing fractions, fl=(1−δ)×f∞ andfh=(1+δ)*f∞, where f∞ is the acid mixing fraction at infinite time. Byappropriate adjustment of the initial pH's of the two liquids to bemixed, the pH's at these two mixing fractions will be the pKa of thebasic indicator and the pKa of the acidic indicator, respectively. Thechemistry underlying DISMT is that of a strong acid/strong base aqueoustitration in the presence of two weak acids, the pH indicators. Thetitration equation is described in Melton et al. and is incorporatedherewith by reference.

Preferably, a colorimetric method comprises image processing ofdigitized images of the whole reactor in combination with imagingprocessing software. Thereby, it is advantageously possible to detect acolor change at a particular location in the reaction chamber veryprecisely, and thus the respective (local) concentration of the tracerand/or indicator, as well as the changes thereof.

Alternatively, the blend time of a reactor can be determined by computersimulations using computational fluid dynamics (CFD) (cf., e.g., Bai etal., 2007. J. Pharm. Sci. 96(11):3072-86, incorporated herein byreference).

Such methods for determining the blend time are described in detail inthe art and were shown to be highly reproducible (cf. e.g. Bai et al.,2007. J. Pharm. Sci. 96(11):3072-86; Cabaret et al. (Mixing TimeAnalysis Using Colorimetric Methods and Image Processing. Industrial &Engineering Chemistry Research, 2007. 46: p.); Paul, E. L.,Atiemo-Obeng, V. A, Kresta, S. M. Handbook of Industrial Mixing. 2004,Hoboken: John Wiley & Sons, Inc.; Manna L. 1997. Comparison betweenphysical and chemical methods for the measurements of blend times. ChemEng J 67:167-173; all incorporated herein by reference).

In the present invention, a method of colorimetric measurement of theblend time is preferably used, wherein the blend time is preferablymeasured by visual inspection, more preferably in a non-intrusivemanner.

In a particularly preferred embodiment, the blend time is experimentallydetermined by a chemical method based on colorimetric measurements.

In a further preferred embodiment, in order to determine the blend timein a reactor (e.g. in a dynamic mixer), the following method derivedfrom Bai et al. may be used: The mixing chamber (reactor chamber) isfilled with an appropriate volume of a basic solution, (preferably NaOH,preferably 0.01-1 mol/L) (solution 1). A pH indicator is added(preferably bromophenole blue or phenolphthalein, preferably in aconcentration of 0.01%). Under stirring (but without flow-through), avolume and molar equivalent of an acid solution (same volume and samemolarity as solution 1) (solution 2) is added to solution 1 and the timeneeded for colour change of the pH indicator is measured. Preferably,the measurement is carried out in at least quintruplicates. (Solution 1and Solution 2 can be interchanged).

In the context of the present invention, a uniformity level(homogeneity) is defined as the endpoint of the mixing process, i.e. thetime point, when blend time is determined. More preferably, the endpointof the mixing process is defined by a uniformity level (homogeneity) ofat least 70%, at least 80%, at least 85%, at least 90% or mostpreferably at least 95%.

To simplify the measurement of the blend time (particularly if shortblend times are measured), a solution increasing the viscosity as e.g.glycerol may be added to solution 1 and/or 2, preferably in aconcentration of from 1 to 90% (v/v), more preferably in a concentrationof from 50 to 80% (v/v), most preferably in a concentration of 75%(v/v). In this set-up, the blend time determined with solutionscomprising glycerol is longer compared to solutions without glycerol andtherefore having a lower viscosity. Thus, it can be concluded that thespecific conditions used for determining the blend time (stirring rate,etc.) resulting in a specific measured blend time (e.g. 5 s or 2 s)result in a shorter blend time, if solutions to be mixed are usedwithout glycerol and therefore with a lower viscosity.

In a specific embodiment of the present invention, the blend time of areactor is determined according to the measurement method described indetail in the following:

The reaction chamber of the reactor is filled completely with a solutionof 75% glycerol/0.01M NaOH/0.01% bromophenol blue in water (solution 1).Under stirring (but without flow-through), a volume of 0.01 M HClcorresponding to 0.01 volume equivalents of solution 2 is added and thetime until the colour has changed from purple to yellow is recordedvisually in quintuplicates.

According to the method of the invention, the reaction between the atleast one RNA in the first liquid composition and the at least onecationic or polycationic compound in the second liquid composition isconducted, i.e. started and preferably completed, within the reactionchamber of the reactor (e.g. a tank reactor). Subsequently, the productliquid composition comprising the nanoparticle comprising the at leastone RNA and the at least one cationic or polycationic compound isrecovered from the reactor in step (d). By conducting the completereaction in the reaction chamber of the reactor under controlledconditions, unwanted side reactions and the formation of unwanted sideproducts can be avoided advantageously, so that a product liquidcomposition is obtained, which comprises the nanoparticle comprising theat least one RNA and the at least one cationic or polycationic compound,which is characterised by uniform average particles size andpolydispersity, reliably under controlled conditions, independent of thescale of production.

The product liquid composition may be recovered by any means known inthe art, for example means as those used to introduce the first andsecond liquid compositions into the reactor, e.g. by pumping, or thelike.

In the case of an open reactor, the product liquid composition may byrecovered through the opening of the reaction chamber, e.g. by suctionpumping, or the like, or by means of passive transport, e.g. a syphoncontrolling the fill level of the reaction chamber, or the like.

In the case of a closed reactor under continuous operation, the productliquid composition may also be recovered from the reactor by means ofpassive transport, e.g. a flow resulting from the pressure build-up inthe reactor caused by a continuous introduction of the first and secondreagent compositions, or the like.

Preferably, the product liquid composition is recovered from the reactorand transferred to a reservoir vessel, which optionally may be held at apredetermined temperature and/or pressure using appropriatetemperature-controlling and pressure-controlling means, respectively,known in the art. The reservoir vessel can be connected to the reactorvia any connection means known in the art, e.g. by means of a tube, ahose, tubing, etc. Materials suitable for the reservoir vessel andconnections means are known in the art, and may be the same as thoseused for the reactor described above.

Alternatively, the product liquid composition may be dividedappropriately and transferred to individual dosage vessels, whichoptionally may be sealed thereafter, or the like, using means fordividing and filling known in the art, optionally maintaining apredetermined temperature and/or pressure using appropriatetemperature-controlling and pressure-controlling means, respectively,known in the art.

Preferably, the at least one reactor comprises at least one mixingmeans. In a preferred embodiment, the reactor comprises at least onedynamic mixing means and/or at least one static mixing means.

In the context of the invention, the term ‘mixing means’ is used for adevice that mixes the content of the reaction chamber of the reactor. Inprinciple, any device known in the art may be used in the presentinvention, as long as the following requirements are provided: Themixing means must allow to thoroughly mix the contents of the reactionchamber of the tank reactor, and that the tank reactor has a blend timeof 5 seconds or less. The mixing means mixes the contents of the reactorwith a blend time of 5 seconds or less. As used herein, the term ‘mixingmeans’ may also refer to a feature or a specific structure of thereactor (e.g. a static mixing means), which allows mixing of the firstand second liquid compositions. In the context of the present invention,a ‘static mixing means’ may also be any feature of a reactor or mixer,which creates a turbulence that results in mixing of the first andsecond liquid composition. A person skilled in the art will readilyknow, which mixing means is appropriately selected for any given reactordesign. Suitable types and designs of mixing means, as well as theirrespective properties in combination with different reactor designs, areknown in the art, for example, as described, e.g., in Steffe J F.,Mixing and Agitation, Chapter 10 “Mixing and Agitation”, pages 287 to304, etc. (Steffe J F. Mixing and Agitation [Internet]. Second Edition.[cited 2013 Jan. 13] Available on the world wide web atpacontrol.com/process-information-book/Mixing%20and%20Agitation%2093851_10.pdf),which is incorporated herein by reference.

In the context of the invention, “mixing” is typically a process thatinvolves manipulation of a heterogeneous physical system with the intentto make it more homogeneous. Mixing is performed to allow mass transferto occur between one or more streams, components or phases. Mixing isfundamentally the evolution in time of spatially dependentconcentrations toward a final homogeneous state.

A mixing means is defined herein as a device or a structure thatprovides mixing of at least two components to be mixed under controlledconditions.

In the method of the invention, two liquids are mixed, namely the firstliquid composition and the second liquid composition. Thereby, thereagents respectively comprised therein are brought into contact forreaction under controlled conditions.

In a preferred embodiment of the present invention, wherein the reactorcomprises at least one mixing means, the blend time of 5 seconds or lesscan be readily maintained in the reactor system and/or adjusted uponchange of other process parameters and/or conditions.

Preferably, the at least one mixing means comprises at least one dynamicmixing means and/or at least one static mixing means. Suitable dynamicmixing means and static mixing means are known in the art (cf. above).

According to a preferred embodiment of the invention, the reactor is adynamic mixer, which preferably comprises at least one dynamic mixingmeans. Dynamic mixers are typically systems with at least one movingelement (or dynamic mixing means), such as a moving stirring means or amoving vessel. A dynamic mixer is any mixer that provides agitation andmixing of the solutions/components to be mixed therein by application ofmechanical forces, for example, agitation by movement of a paddle orblade connected to an external power source, such as a motor or amagnetic stirrer. In such instances, the mixing of thesolutions/components to be mixed is accomplished by the rotation of thepaddle or blade, which creates the turbulence necessary for mixing. Inanother embodiment agitation and mixing is performed by a shaker,vortexer or the like. The dynamic mixers appropriate for use in themethods and apparatuses disclosed herein should comprise materials(e.g., stainless steel, glass, polyethylene) which are inert to thereaction conditions, do not react with the solutions/components to bemixed and/or do not influence the components to be mixed. Such materialsare used in the areas of the mixer where the solutions/components to bemixed contact the mixer.

Dynamic mixers are commercially available and may be purchased, forexample, from Knauer (Germany, Dynamic mixing chamber order #A1174),Sielc (USA, Dynamic mixer order #DMP-1031050) or Gilson (USA, Dynamicmixer 811 D, order #LT3634D).

In the context of the present invention, a mixing means is preferablyused, which allows for sufficient mixing of the components as definedherein, preferably without exerting excessive mechanical stress (such asshear stress) on said components. It is believed that the stability ofthe at least one RNA, preferably a long chain RNA or a mRNA as definedherein, the cationic or polycationic compound and/or of the nanoparticleis increased by using appropriate (dynamic or static) mixing means. Inparticular, mixing means that are known to induce mechanical stress onthe components to be mixed are preferably avoided according to thepresent invention. For example, a mixing means as used herein doespreferably not shake and/or agitate (such as when using a ‘Vortex’mixer) the at least one reactor, the first liquid composition and/orsecond liquid composition. According to one embodiment, the first liquidcomposition and the second liquid composition are preferably mixedwithout shaking and/or agitating the at least one reactor or the liquidcompositions, respectively. More preferably, the term ‘agitating’ asused with respect to the inventive method refers to a process that doesnot involve mechanical stress, such as shaking. The term ‘agitating’preferably refers to mixing of the components by turbulences that arenot caused by shaking or vibrating of the reactor or the first and/orsecond liquid composition. It is particularly preferred that the firstliquid composition and the second liquid composition are mixed withoutthe use of a ‘Vortex’ mixer, or the like.

In another preferred embodiment of the invention, the reactor is astatic mixer, which preferably comprises at least one static mixingmeans. Static mixers are typically systems, in which the mixing processis typically initiated by using the hydrodynamic energy of a fluid(liquid/gas) passing through a cavity, such as a pipe or canal, withfixed fittings. Static mixers are stationary systems, through which thematerial is flowing. In one embodiment, the mixing effect is exclusivelyachieved by vortexing the flow with the aid of at least one staticmixing means. As used herein, a static mixing means is typically anintegral element of the reactor, which is not moving. Preferably, the atleast one static mixing means comprises at least one static element,preferably at least one static element positioned within a flow path ofthe first liquid composition and/or the second liquid composition.Moreover, as used herein, a ‘static mixing means’ may also be anyfeature, such as the injection of one liquid into another liquid stream,which results in a turbulence that leads to mixing of the liquids.Alternatively, the system may further comprise dynamic mixing means,which contribute to the mixing effect. Precondition for the applicationof static mixers is the pumpability of the original materials. A staticmixer is preferably a mixer that does not rely on mechanical agitation,shaking or stirring by a mechanical device in the mixer. Rather, astatic mixer relies on static elements, such as walls, channels,capillaries, barriers, offset plates or protrusions such as rods ornubs, or the like, any of which may comprise holes or openings, or beoffset from one another, or the like, to direct the flow path in a wayto provide flow turbulence and mixing of the solutions/components to bemixed as they proceed through the mixer. In such a case, the flow rateand pressure is determined by a number of factors, including forexample, the flow rate of each solution being introduced, the size anddiameter of the connectors and inlet and outlet openings in eachcomponent, the size and shape of the chamber, the volume to surface arearatio of the mixer, the number of flow paths and flow path diversions inthe mixer, and the like. The mixer may comprise a tee configuration or aserpentine configuration.

In a specific embodiment, the reactor is a static mixer as describedherein (e.g. a tube reactor) and comprises an elongate cavity, throughwhich the first liquid composition and/or the second liquid compositionflow. In this context, the elongate cavity may have any shape ordimension, as long as the first and the second liquid compositions aremixed with a blend time of 5 seconds or less. Preferably, the cavity mayhave the shape of a cylinder, a tube, a pipe, a capillary or a hose.

In a further preferred embodiment, the reactor is a static mixer,wherein one liquid composition selected from the first liquidcomposition and the second liquid composition flows through the reactorand the respective other liquid composition is injected into the liquidthat flows through the reactor, wherein the respective other liquidcomposition is injected in the reactor or upstream of the reactor. Theliquid is preferably injected at the center of the liquid that flowsthrough the reactor.

A static mixer is typically a precision device, which is preferablyengineered for the continuous mixing of fluid materials. One example ofa design of a static mixer is the plate-type mixer. Another example is astatic mixer comprising at least one static mixing means contained in acylindrical (tube) or squared housing. Mixer size can vary from about 6mm to 6 meters diameter. Typical construction materials for static mixercomponents include stainless steel, polypropylene, PTFE (Teflon®), PVDF,PVC, CPVC and polyacetal.

Static mixers can use flow division or radial mixing for mixing.

Flow division: In laminar flow, a processed material is divided at theleading edge of each element of the mixer and follows the channelscreated by the element shape.

Radial mixing: In either turbulent flow or laminar flow, rotationalcirculation of a processed material around its own hydraulic center ineach channel of the mixer causes radial mixing of the material.Processed material is intermixed to reduce or eliminate radial gradientsin temperature, velocity and material composition.

Commericial suppliers of static mixers are, for example, Sulzer(Switzerland, e.g. SMX mixing elements), Nordson (USA) orChromatographie Handel Müller (Germany, order #760092).

A mixing means comprised in the reactor used in the method of thepresent invention may comprise both dynamic and static mixing elements,e.g. one or more impeller blades in combination with baffles, or thelike.

Preferably, the at least one mixing means comprises at least onestirring means. Suitable stirring means are known in the art (cf.above). The stirring means may be any known stirrer design known in theart, e.g. impeller blades or paddles mounted on an axis driven by anexternal motor, a stir bar driven by a magnetic force provided by anexternal rotation device, or the like.

A preferred embodiment of a stirring means is a magnetic stirrer,wherein the reaction chamber comprises a stirring bar (such as amagnetic stirrer), which is rotated by a magnetic force provided by anexternal device (motor). The dimension of the stirring bar can beappropriately selected in accordance with the reactor design, size, etc.Preferable examples of stirring bars comprise a core made of amagnetisable material (e.g. iron, or the like) which is coated with aninert material (usually PTFE, Teflon®, or the like). Another preferredembodiment of a stirring means is an impeller, which is known in theart.

Preferably, the rotational speed of the stirring means can be controlledby controlling the respective speed of the drive, either step-wise orinfinitely variable.

Preferably, the stirring means can be operated at a stirring rate of atleast 300 rpm, preferably of at least 500 rpm, and more preferably of atleast 1000 rpm.

In a preferred embodiment of the present invention, wherein the mixingmeans comprises at least one stirring means, the rotational speed ofwhich can be controlled, the blend time of 5 seconds or less can bereadily maintained in the reactor system and/or adjusted upon change ofother process parameters and/or conditions.

In a preferred embodiment, the reactor, preferably a tank reactor,comprises at least one stirring means, preferably a single stirringmeans. However, the reactor can also comprise two or more stirringmeans, e.g. in the form of two impellers mounted on opposite sites ofthe reaction chamber (e.g. at the top and bottom thereof), which may beoperated in the same or the opposite rotational direction, at the sameor different rotational speeds, and the like.

According to the method of the present invention, the first liquidcomposition comprising at least one RNA and the second liquidcomposition comprising at least one cationic or polycationic compoundare introduced into at least one reactor.

Preferably, the method of the invention is conducted continuously ordiscontinuously (non-continuously).

For a discontinuous operation, for example, the method is typicallycarried out using predetermined batches, e.g. by introducingpredetermined volumes of both the first liquid composition and thesecond liquid composition into the reactor, and, after completion of thereaction, recovering the product liquid composition preferably having avolume corresponding to the combined volumes of the first and secondliquid compositions from the reactor. This procedure is then repeated.

A discontinuous operation is preferably carried out in a reactor adaptedfor discontinuous operation (also referred to as “Non-continuousReactor”). In the context of the invention, a “Non-continuous Reactor”is a reactor, preferably a tank reactor, that controls that at least twoliquid compositions (solutions), which are to be mixed and reacted, mix,contact, and react in the reactor under controlled conditions, and thatthe product liquid composition remains in the reactor where the mixingand reaction occurs, until recovered in a subsequent step. Typically,the total liquid volume in the reactor increases and is limited by thereaction chamber volume. An example of a Non-continuous Reactor isReactor II shown in FIG. 2 .

For a continuous operation, for example, both the first liquidcomposition and the second liquid composition are continuouslyintroduced into the reactor, and the product liquid composition iscontinuously recovered from the reactor. The product liquid compositionis recovered with a flow rate that corresponds to the combined flowrates of the first and second liquid compositions (also referred to as“total flow rate”). Preferably, the flow rates of both the first liquidcomposition and the second liquid composition are identical, so that thetotal flow rate of the product liquid composition is the sum of the flowrate of the first liquid composition and the second liquid composition,respectively. In this case, the means for recovering the product liquidcomposition from the reactor must allow for the larger flow rate andvolume.

According to another embodiment, a continuous operation is carried outin a reactor, preferably in a reactor adapted for flow-through operation(also referred to as “Continuous Reactor”), such as a flow-throughreactor with an elongate cavity, e.g. a tube reactor, a tank reactoradapted for flow-through operation, or any other reactor suitable forflow-through operation. In the context of the invention, a “ContinuousReactor” is a reactor that simultaneously controls that at least twoliquid compositions (solutions) to be mixed and reacted mix, contact,and react in the reactor under controlled conditions, and that productliquid composition exits from the continuous reactor at a controlledrate. Preferably, the total liquid volume in the mixing device ismaintained constant. A continuous reactor allows the flexible scale-upof the reaction process. Therefore, the use of a continuous reactor isparticularly preferred. A continuous reactor is also referred to hereinas “Flow-Through Reactor”. Preferred examples of a continuous reactorare a closed tank reactor in continuous operation or a static mixer,such as a flow-through reactor with an elongate cavity, e.g. a tubereactor or an injection mixer. Examples of a continuous reactor areReactors III to VII as shown in FIGS. 4, 5, 6, 10, 11, 12, 13 and 14 .

Preferably, the first and the second liquid compositions are introducedinto the at least one reactor successively or concurrently.

In one preferred embodiment of the method of the present invention, thefirst and the second liquid compositions are introduced into the atleast one reactor successively. For a successive introduction, forexample, one of the first and second liquid compositions are firstintroduced into the reactor, then the other liquid composition is addedthereto, either stepwise or continuously. A successive introductionmethod is preferably carried out in combination with a reactor, such asa tank reactor, in discontinuous (batch) operation as described above(“Non-continuous Reactor”).

In an alternative preferred embodiment of the present invention, boththe first liquid composition and the second liquid composition areconcurrently (simultaneously) introduced into the at least one reactor.For a concurrent introduction, for example, both the first liquidcomposition and the second liquid composition are introduced into thereactor at the same time (simultaneously), preferably in amounts toprovide the predetermined ratio of products to be reacted as definedherein, especially preferred in equal volumes of the first and secondliquid compositions (e.g. by using equal flow rates). A concurrentintroduction method is preferably carried out in combination with aflow-through reactor, such as a tube reactor or a flow-through tankreactor in continuous operation as described above (“ContinuousReactor”).

Especially preferred, both the first liquid composition and the secondliquid composition are concurrently introduced into the at least onereactor with the same flow rate.

In an especially preferred embodiment of the invention, the method ofthe invention is conducted continuously with concurrent introduction ofthe first and second liquid compositions, the at least one reactor is aflow-through tank reactor, and the at least one mixing means is adynamic mixing means, preferably a stirring means. Alternatively, themethod of the invention is conducted continuously with concurrentintroduction of the first and second liquid compositions, wherein the atleast one reactor is a flow-through reactor, preferably a static mixer,more preferably a static mixer as defined herein.

In another preferred embodiment of the invention, the method of theinvention is conducted discontinuously with successive introduction ofthe first and second liquid compositions, the at least one reactor is anon-continuous tank reactor, and the at least one mixing means is adynamic mixing means, preferably a stirring means.

In another preferred embodiment of the method according to the presentinvention, the first liquid composition comprising at least one RNA andthe second liquid composition comprising at least one cationic orpolycationic compound are continuously introduced into at least oneflow-through reactor, in which the first liquid composition and thesecond liquid composition are mixed and preferably simultaneouslyreacted with each other, wherein the flow-through reactor comprises atleast one static mixing means. Preferably, the flow-through reactor is astatic mixer as defined herein (e.g. a tube reactor) and comprises anelongate cavity. According to that embodiment, the flow-through reactor,which is a static mixer, may optionally comprise one or more dynamicmixing means as defined herein.

In the context of the invention, a “flow-through reactor” is a typicallya continuous reactor wherein the first liquid composition and/or thesecond liquid composition is continuously flowing through the reactionchamber, which preferably comprises or consists of an elongate cavity.In a preferred embodiment, the flow-through reactor is a static mixer asdescribed herein, which preferably comprises an elongate cavity (e.g. atube reactor). In the context of the present invention, the elongatecavity of a flow-through reactor may have any shape or dimension, aslong as the first and the second liquid compositions are mixed with ablend time of 5 seconds or less. Preferably, the elongate cavity mayhave the shape of a cylinder, a tube, a pipe, a capillary or a hose. Ina preferred embodiment, the flow-through reactor has a straight shape oris bent (e.g. in helices or any other type of curves). The cross-sectionof such a reactor may also be of any shape, such as circular, oval,rectangular, polygonal or any arbitrary shape. The diameter of thereactor may be constant over its length or vary. In a preferredembodiment, the flow-through reactor, preferably the inner walls of theflow-through reactor, comprise at least one static mixing means asdescribed herein, such as a protrusion, which preferably represents anobstacle for the liquid passing through the reactor. Examples of suchreactors are known in the art and may also be referred to as a “tubereactor”, “tubular reactor” or “pipe reactor”, or the like. For example,a flow-through reactor may have the form of a tube, wherein the reagentsare continuously introduced into one end thereof, while the products arecontinuously recovered from the other end thereof. In a preferredembodiment, a flow-through reactor, preferably with an elongate cavity,comprises a reaction space (reaction chamber) having a smaller volume,sometimes referred to as “reaction zone”. The reaction zone of the tubereactor may optionally comprise appropriate temperature-controllingand/or pressure-controlling means, respectively, to control apredetermined temperature and/or pressure within. Suitabletemperature-controlling and pressure-controlling means are known in theart.

A flow-through reactor, preferably a flow-through reactor with anelongate cavity, may have any appropriate length and diameter.

In a preferred embodiment, the flow-through reactor, preferably aflow-through reactor with an elongate cavity, has a length of at least 1cm, 2 cm, 4 cm, 10 cm, or 100 cm.

In a further preferred embodiment, the flow-through reactor, preferablya flow-through reactor with an elongate cavity, has a diameter of atleast 1 mm, 3 mm, 5 mm, or 10 mm.

The flow-through reactor, preferably the flow-through reactor with anelongate cavity, may be of any type and design known in the art, as longas the reactor is provided with a design allowing the mixing andreacting of the first and second liquid compositions under controlledconditions, preferably the simultaneous mixing and reacting of the firstand second liquid compositions under controlled conditions. Preferably,the reactor design provides a reaction space (“reaction zone”), whereinthe reaction between the at least one RNA and the at least one cationicor polycationic is completed under thoroughly controlled conditions.After completion of the reaction, preferably only the product liquidcomposition comprising the product nanoparticle comprising the at leastone RNA and the at least one cationic or polycationic compound leavesthe reaction zone of the reactor. Thereby, the method according to thepresent invention produces the product nanoparticle, which has uniformaverage particles size and polydispersity, reliably, under controlledconditions.

In a preferred embodiment, the flow-through reactor is a static mixer,which comprises at least one helical element. The first and secondliquid compositions are introduced into one end of the reactor, and themixing means comprising at least one helical element provides theturbulence, by which the two streams of reagent solutions are thoroughlymixed, and the product liquid composition leaving the reaction zone isrecovered from the other end of the reactor. An example of aflow-through reactor, preferably a static mixer, comprising a mixingmeans comprising helical elements is shown in FIG. 6 . It was found thatthe method of the invention may be advantageously carried out using areactor comprising helical elements.

In a preferred embodiment, the first liquid composition and the secondliquid composition are introduced into the reactor, preferably a staticmixer as described herein, via separate openings (cf. FIG. 6 ).Alternatively, the first liquid composition and the second liquidcomposition may be unified (e.g. by a tee piece) prior to introductioninto the reactor (cf. FIG. 10 ). Separate introduction of the first andthe second composition is particularly preferred.

In another preferred embodiment of the invention, the turbulence formixing is provided by introducing a stream of one liquid compositionwith an angle α into a stream of the other liquid composition (alsoreferred to as “injector”). According to that embodiment, the reactor ispreferably a static mixer, wherein one liquid composition selected fromthe first liquid composition and the second liquid composition flowsthrough the reactor and the respective other liquid composition isinjected into the liquid that flows through the reactor, wherein therespective other liquid composition is injected in the reactor orupstream of the reactor. Preferably, the reactor comprises an inlet portarranged at a side wall of the reactor, through which one of the reagentliquid compositions is introduced into an elongate cavity of thereactor, for example into the tube of a tube reactor, through which theother reagent liquid composition flows. The inlet port is preferablycharacterized by a diameter, which is inferior to the diameter of thereactor. According to a preferred embodiment, the ratio of the inletport diameter to the reactor diameter is in a range from 1:2 to 1:20,preferably from 1:3 to 1:18, more preferably from 1:5 to 1:15. Forexample, the diameter of the inlet port may be in a range from 0.2 mm to2 mm, more preferably from 0.5 to 1 mm. The angle α is defined as theangle, with which the two reagent streams meet in the reactor. Forexample, the angle α may be determined by the angle between thelongitudinal direction of the main body of the reactor, wherein thefirst liquid composition flows, and the inlet port for the second liquidcomposition, or angle between the inlet ports for the two liquidcompositions, or the like. Preferably, the angle α is from 10 to 90°,further preferred from 20 to 80°, and especially preferred about 30°.

An example of a reactor comprising an injector is shown in FIGS. 11 and12 . It was found that the method of the invention may be advantageouslycarried out using a reactor, preferably a tube reactor, comprising aninjector.

Preferably, the first liquid composition and the second liquidcompositions each are introduced into the at least one reactor,preferably into a flow-through reactor, more preferably into having anelongate cavity such as a tube reactor, with a flow rate of 10 ml/minuteor more, preferably with a flow rate of 50 ml/minute or more, and morepreferably with a flow rate of 125 ml/minute or more. Preferably, thetotal flow rate is 20 ml/minute or more, preferably 100 ml/minute ormore, and more preferably 250 ml/minute or more, and more preferably 500ml/minute or more, and more preferably 750 ml/minute or more, and mostpreferably 1000 ml/minute and more. It was found that the method of theinvention provides excellent results with these flow rates.

The method of the present invention is a method for producing a liquidcomposition comprising a nanoparticle comprising at least one RNA and atleast one cationic or polycationic compound.

It is assumed that the individual components of the nanoparticle aredirectly or indirectly bound to each other by means of electrostaticforces, hydrogen-bridges, or the like. A nanoparticle once formed isassumed to be a stable entity because of cooperative effects caused bythe polymeric nature of the components.

Advantageously, the nanoparticles obtained by the method according tothe present invention are characterised by having an uniform averageparticle size and particle size distribution, preferably as defined inthe following.

In the context of the invention, a nanoparticle is a particle,preferably a solid particle which is characterised by having an averageparticle size and a particle size distribution. In the art, severalmethods to measure the average particle size of a nanoparticle areknown.

In the context of the invention, the average size of the nanoparticlesis measured by dynamic light scattering, whereby the particle size isrepresented as hydrodynamic diameter of a spherical particle. Theso-measured average particle size is represented as Z-average in nm.

The measurements can readily be carried out using a suitable instrument.An example of a commercially available instrument is a Zetasizer NanoZS, which is available from Malvern Instruments, Malvern, U.K. Themeasurement is preferably carried out at 25° C., preferably using ascattering angle of 173°. Further details of the measurement of theaverage particle size of a nanoparticle are described in Example 2.

In a preferred embodiment, the nanoparticle comprising at least one RNAand at least one cationic or polycationic compound obtained by themethod according to the present invention has a particle size of 500 nmor less, more preferably from 50 to 500 nm, more preferably from 50 to300 nm, more preferably from 50 to 200 nm, more preferably from 50 to150 nm. Preferably, the nanoparticles comprising at least one RNA and atleast one cationic or polycationic compound obtained by the methodaccording to the present invention have an average particle size of 500nm or less, more preferably from 50 to 500 nm, more preferably from 50to 300 nm, more preferably from 50 to 200 nm, more preferably from 50 to150 nm.

As a measure for the particle size distribution, the polydispersity andpolydispersity index (PDI) can be used.

Polydispersity describes the width of a population distribution. Itdescribes the degree of heterogeneity of a population. In this context,polydispersity represents the width of the particle size distribution ofthe nanoparticles present in a liquid composition, particularly thewidth of the particle size distribution of the product RNA-comprisingnanoparticles.

The polydispersity index (PDI) is a dimensionless measure of thebroadness of the size distribution in nanoparticle samples.Polydispersity index values below 0.1 indicate a monomodal distribution,while a polydispersity index over 0.5 indicates a broad distribution ofparticle sizes. Analytical instruments and methods for the calculationof these parameters used for size measurements have been described (e.g.Zetasizer Nano Series; User Manual MAN0314 Issuer 1.1 February 2004).

The measurements of the PDI can readily be carried out using a suitableinstrument. An example of a commercially available instrument is aZetasizer Nano ZS, which is available from Malvern Instruments, Malvern,U.K. Further details of the measurement of the PDI of a nanoparticle aredescribed in Example 2.

The polydispersity index (PDI), sometimes referred to as heterogeneityindex, or simply dispersity (Ð), is a measure of the distribution ofmolecular mass in a given nanoparticle sample. The PDI calculated is theweight-average molecular weight (Mw) divided by the number-averagemolecular weight (Mn):PDI=Mw/Mn,

where Mw is the weight average molecular weight and Mn is the numberaverage molecular weight. The PDI indicates the distribution ofindividual molecular masses in a batch of nanoparticles.

Preferably, the nanoparticles comprising at least one RNA and at leastone cationic or polycationic compound have a polydispersity index offrom 0.05 to 0.50, preferably of from 0.05 to 0.40, and more preferablyof from 0.10 to 0.30.

This represents a monomodal to narrow particle size distribution, whichis advantageously obtained in RNA-comprising nanoparticles with themethod according to the present invention.

In the context of the present invention, particularly preferred areRNA-comprising nanoparticles with a PDI of 0.5 or below, and morepreferred with a PDI of 0.4 or below, and most preferred with a PDI of0.3 or below.

Preferably, the product liquid composition comprising a nanoparticlecomprising at least one RNA and at least one cationic or polycationiccompound obtained by the method of the invention is a dispersion ofnanoparticles comprising at least one RNA and at least one cationic orpolycationic compound, preferably a stable colloidal dispersion inwater.

A stable colloidal dispersion is a dispersion of nanoparticles, whichdoes not deteriorate with time, even after long-time storage underappropriate conditions.

Advantageously, the product liquid composition comprising anRNA-comprising nanoparticle obtained according to the method of thepresent invention comprises uniform RNA-comprising nanoparticles in highquality and excellent yields, without comprising significant amounts ofunwanted side products, which lead to turbidity (precipitates) in theproduct compositions and the ultimate deterioration thereof. Inparticular, the method according to the present invention allows toproduce RNA-comprising nanoparticles in a quality sufficient andreliable for pharmaceutical applications, even in a large scaleproduction. The method of the invention further allows obtainingexcellent yields.

In order to assess the quality of the product liquid compositioncomprising the RNA-comprising nanoparticle obtained by the methodaccording to the present invention, the composition is preferablyinspected visually, more preferably by using image processingtechniques. Product compositions having the desired quality aretypically clear. An inferior quality of the product composition isvisible, for example, as turbidity within the composition, whereinincreasing turbidity is correlated with decreasing product quality andstability, eventually resulting in the formation of precipitates.

In order to obtain a numerical value for assessing the product qualityby means of its clarity, the absorption of the product liquidcomposition can also be measured. Preferably, the absorption of theproduct liquid composition comprising the compound comprising at leastone RNA and at least one cationic or polycationic compound is measureddirectly after the production thereof. This measurement of absorptionmay be preferably and advantageously carried out in-line in a continuousproduction process.

In the context of the invention, absorption measurements are carried outat 350 nm. Further details of the measurement of the absorption at 350nm of a RNA-comprising nanoparticle-containing product composition aredescribed in Example 2.

Preferably, the product liquid composition has an absorption at 350 nmof 0.5 or less, of 0.4 or less, of 0.3 or less, more preferably of 0.2or less and most preferably of 0.15 or less.

Additionally or alternatively, the quality of the product liquidcomposition comprising the compound comprising at least one RNA and atleast one cationic or polycationic compound is preferably assessed bymeasuring its turbidity. For example, the turbidity may be measured at860 nm with a detecting angle of 90° using commercially availableinstruments and methods known in the art. An example for a commerciallyavailable instrument is a NEPHLA turbidimeter, available from Dr. Lange,Düsseldorf, Germany. The system is calibrated with formazin as standardand the results were given in formazin nephelometric units (FNU). Thismethod and other methods useful for measuring turbidity/clarity areknown in the art and are e.g. described in EUROPEAN PHARMACOPOEIA 5.0,2.2.1. Clarity and degree of opalescence of liquids and ISO7027:1999-Water quality.

Further details of the measurement of the turbidity (FNU) of aRNA-comprising nanoparticle-containing product composition are describedin Example 2.

Preferably, the product liquid composition comprising the compoundcomprising at least one RNA and at least one cationic or polycationiccompound has a turbidity of 100 FNU or less.

In a preferred embodiment, the method according to the invention furthercomprises a step (e) of isolating or concentrating the compound and/ornanoparticles comprising at least one RNA and at least one cationic orpolycationic compound from the product liquid composition comprising thenanoparticle comprising at least one RNA and at least one cationic orpolycationic compound.

Suitable methods for isolating the nanoparticle from the product liquidcomposition are lyophilisation, spray-drying, spray-freeze drying, orthe like.

Preferably, step (e) comprises a drying step selected from the groupconsisting of lyophilisation, spray-drying, or spray-freeze drying.

In a further aspect, the present invention provides a liquid compositioncomprising a nanoparticle comprising at least one RNA and at least onecationic or polycationic compound which is obtainable by the methodaccording to the present invention, and/or the nanoparticle comprisingat least one RNA and at least one cationic or polycationic compoundwhich is obtainable and preferably isolated by the method according tothe present invention.

In yet another aspect, the present invention concerns the use of theinventive method in the manufacture of a medicament or a vaccine,preferably a medicament or a vaccine for use in the treatment orprophylaxis of a disorder or a disease. In the context of the presentinvention, the disorder or the disease is preferably selected from thegroup consisting of cancer or tumor diseases, infectious diseases,preferably viral, bacterial or protozoological infectious diseases,autoimmune diseases, allergies or allergic diseases, monogeneticdiseases, i.e. (hereditary) diseases, or genetic diseases in general,diseases which have a genetic inherited background and which aretypically caused by a single gene defect and are inherited according toMendel's laws, cardiovascular diseases and neuronal diseases.

The present invention further relates to the medical use of the liquidcomposition comprising the nanoparticle comprising at least one RNA andat least one cationic or polycationic compound, which is obtainable bythe method according to the present invention. Moreover, the presentinvention relates to the medical use of the nanoparticle comprising atleast one RNA and at least one cationic or polycationic compound, whichis obtainable by the the method according to the present invention. In apreferred embodiment, the invention provides the liquid compositioncomprising the nanoparticle comprising at least one RNA and at least onecationic or polycationic compound, which is obtainable by the methodaccording to the present invention or the nanoparticle comprising atleast one RNA and at least one cationic or polycationic compound, whichis obtainable by the method according to the present invention, for usein the treatment or prophylaxis of a disease or disorder as definedherein.

Furthermore, the present invention relates to a method of treatment,wherein the method comprises administering to a subject in need thereofthe liquid composition comprising the nanoparticle comprising at leastone RNA and at least one cationic or polycationic compound, or thenanoparticle comprising at least one RNA and at least one cationic orpolycationic compound, which are obtainable by the method according tothe present invention. Preferably, the liquid composition or thenanoparticle is administered to a subject via any suitableadministration route, preferably in a safe and effective amount.

Preferably and advantageously, the nanoparticle comprising at least oneRNA and at least one cationic or polycationic compound, or the productliquid composition comprising the nanoparticle comprising at least oneRNA and at least one cationic or polycationic compound are for use in apharmaceutical composition suitable for RNA therapy.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising the nanoparticle comprising at least one RNA andat least one cationic or polycationic compound, or the product liquidcomposition comprising the nanoparticle comprising at least one RNA andat least one cationic or polycationic compound, obtainable by the methodaccording to the present invention. Preferably, the pharmaceuticalcomposition is for use in the treatment or prophylaxis of a disease ordisorder as defined herein.

Furthermore, the invention is preferably described by the followingitems:

1. A method for producing a liquid composition comprising a nanoparticlecomprising at least one long-chain RNA and at least one cationic orpolycationic compound,

wherein the method comprises the steps of:

(a) providing a first liquid composition comprising at least one RNA,

(b) providing a second liquid composition comprising at least onecationic or polycationic compound,

(c) introducing the first liquid composition and the second liquidcomposition into at least one reactor, wherein the first liquidcomposition and the second liquid composition are mixed with a blendtime of 5 seconds or less, and

(d) recovering the product liquid composition comprising thenanoparticle comprising the at least one RNA and the at least onecationic or polycationic compound from the reactor.

2. The method according to item 1, wherein the at least one RNA isselected from the group consisting of a long-chain RNA, a coding RNA, anon-coding RNA, a single-stranded RNA, a double stranded RNA, a linearRNA, a circular RNA (circRNA), a messenger RNA (mRNA), an RNAoligonucleotide, an siRNA, an miRNA, an shRNA, an antisense RNA, ariboswitch, an immunostimulating RNA (isRNA), a ribozyme, an aptamer, aribosomal RNA (rRNA), a transfer RNA (tRNA), a self-replicating RNA(replicon RNA), a CRISPR/Cas9 guide RNA, a small nuclear RNA (snRNA), asmall nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), a retroviralRNA, or a viral RNA (vRNA).

3. The method according to item 1 or 2, wherein the at least one RNA isa long-chain RNA comprising from 100 to 50000 nucleotides, preferablyfrom 200 to 15000 nucleotides, more preferably from 300 to 10000nucleotides, and most preferably from 400 to 7000 nucleotides.

4. The method according to any one of items 1 to 3, wherein the at leastone RNA is not an siRNA.

5. The method according to any one of the preceding items, wherein theat least one RNA is an mRNA.

6. The method according to any one of the preceding items, wherein thenanoparticle comprising at least one RNA and at least one cationic orpolycationic compound has a particle size of 300 nm or less, preferablyof from 50 to 200 nm, and more preferably from 50 to 150 nm.

7. The method according to any one of the preceding items, wherein thenanoparticle comprising at least one RNA and at least one cationic orpolycationic compound has a polydispersity index in a range from 0.05 to0.50, preferably of from 0.05 to 0.40, and more preferably of from 0.05to 0.3.

8. The method according to any one of the preceding items, wherein theproduct liquid composition comprising the nanoparticle comprising atleast one RNA and at least one cationic or polycationic compound is adispersion of the nanoparticle comprising at least one RNA and at leastone cationic or polycationic compound, preferably a stable colloidaldispersion in water.

9. The method according to any one of the preceding items, wherein theproduct liquid composition comprising the nanoparticle comprising atleast one RNA and at least one cationic or polycationic compound hasabsorption at 350 nm of 0.5 or less.

10. The method according to any one of the preceding items, wherein theproduct liquid composition comprising the nanoparticle comprising atleast one RNA and at least one cationic or polycationic compound has aturbidity of 100 FNU or less.

11. The method according to any one of the preceding items, wherein thefirst liquid composition comprises RNA in a concentration of from 0.1 to20 g/L, preferably from 0.5 to 10 g/L, and more preferably from 0.5 to 7g/L.

12. The method according to any one of the preceding items, wherein theat least one cationic or polycationic compound is selected from thegroup consisting of a cationic or polycationic peptide, a cationic orpolycationic protein, a cationic or polycationic polyamino acid, acationic or polycationic carbohydrate, a cationic or polycationicsynthetic polymer, a cationic or polycationic small synthetic organicmolecule, an inorganic multivalent cation, a cationic or polycationiclipid, a cationic or polycationic polyamine compound, and a cationic orpolycationic polyimine compound.

13. The method according to any one of the preceding items, wherein theat least one cationic or polycationic compound is a cationic orpolycationic peptide or protein.

14. The method according to any one of the preceding items, wherein theat least one cationic or polycationic compound is selected from thegroup consisting of protamine, nucleoline, spermine or spermidine,poly-L-lysine (PLL), basic polypeptides, poly-arginine, oligoarginines,cell penetrating peptides (CPPs), HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs),PpT620, proline-rich peptides, arginine-rich peptides, lysine-richpeptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, and histones.

15. The method according to any one of the preceding items, wherein thesecond liquid composition comprises a cationic or polycationic compoundin a concentration in a range from 0.05 to 10.00 g/L, preferably from0.10 to 5.00 g/L or, more preferably, from 0.10 to 1.00 g/L.

16. The method according to any one of the preceding items, wherein thefirst liquid composition and/or the second liquid composition furthercomprise at least one compound selected from a salt or a lyoprotectant.

17. The method according to any one of the preceding items, wherein thefirst liquid composition and/or the second liquid composition compriseat least one salt selected from the group consisting of NaCl, KCl, LiCl,MgCl₂, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, Na₃PO₄, KI, KBr, K₂CO₃, KHCO₃,K₃PO₄, K₂SO₄, CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂, and Ca₃(PO₄)₂.

18. The method according to any one of the preceding items, wherein thefirst liquid composition and/or the second liquid composition compriseat least one cation selected from the group consisting of Na⁺, K⁺, Li⁺,Mg²⁺, Ca²⁺, and Ba²⁺ and/or at least one anion selected from the groupconsisting of Cl⁻, CO₃ ²⁻, PO₄ ³⁻ and SO₄ ²⁻.

19. The method according to item 17, wherein the concentration of thecation in the first liquid composition and/or the second liquidcomposition is up to 50 mM, preferably of from 0.001 to 50 mM, morepreferably from 3 to 30 mM.

20. The method according to any one of items 18 or 19, wherein the ratioof cation to RNA in the first liquid composition is from 3 to 30 mmolcation/g RNA, preferably from 4 to 23 mmol cation/g RNA, and mostpreferably from 5 to 10 mmol cation/g RNA.

21. The method according to any one of items 18 to 20, wherein theconcentration of the anion in the first liquid composition and/or secondliquid composition is 23 mM or less, more preferably from 1.0 to 23.0mM, and especially preferred from 1.0 to 10.0 mM.

22. The method according to any one of the preceding items, wherein thelyoprotectant is selected from the group consisting of sucrose, mannose,trehalose, mannitol, polyvinylpyrrolidone, glucose, fructose, Ficoll 70.

23. The method according to item 22, wherein the concentration of thelyoprotectant in the first liquid composition and/or the second liquidcomposition is in a range from 0.01% (w/w) to 40% (w/w), preferably from1% (w/w) to 20% (w/w).

24. The method according to any one of the preceding items, wherein thefirst liquid composition and/or the second liquid composition compriseswater.

25. The method according to any one of the preceding items, wherein theblend time is experimentally determined.

26. The method according to any one of the preceding items, wherein theblend time is determined by using a method selected from a colorimetricmethod, a method based on conductivity measurements, and a chemicalmethod.

27. The method according to any one of the preceding items, wherein theblend time is determined by using a chemical method on the basis of anacid-base-type reaction using colorimetric detection.

28. The method according to any one of items 1 to 24, wherein the blendtime is determined by computer simulations using computational fluiddynamics (CFD).

29. The method according to any one of the preceding items, wherein thefirst liquid composition and the second liquid composition are added tothe at least one reactor so that the at least one cationic orpolycationic compound and the at least one RNA are present in thereactor with an N/P-ratio in a range from 0.1 to 10, preferably from 0.3to 4, and even more preferably from 0.3 to 0.9.

30. The method according to any one of the preceding items, wherein theat least one reactor has a volume in a range from 1 ml to 10 l,preferably from 1 ml to 5 l, more preferably from 1 ml to 500 ml.

31. The method according to any one of the preceding items, wherein theat least one reactor comprises at least one mixing means.

32. The method according to item 31, wherein the at least one mixingmeans comprises at least one dynamic mixing means and/or at least onestatic mixing means.

33. The method according to any one of the preceding items, wherein thereactor is a dynamic mixer comprising at least one dynamic mixing means.

34. The method according to any one of the preceding items, wherein thefirst liquid composition and the second liquid composition are mixedwithout shaking and/or agitating the at least one reactor or the firstand second liquid compositions, respectively.

35. The method according to any one of items 32 to 34, wherein the atleast one dynamic mixing means comprises at least one stirring means.

36. The method according to item 35, wherein the at least one stirringmeans can be operated at a stirring rate of at least 300 rpm, preferablyof at least 500 rpm, and more preferably of at least 1000 rpm.

37. The method according to any one of items 1 to 33, wherein thereactor is a static mixer.

38. The method according to item 37, wherein the static mixer comprisesat least one static mixing means, which comprises at least one staticelement, preferably at least one static element positioned within a flowpath of the first liquid composition and/or the second liquidcomposition.

39. The method according to item 37 or 38, wherein the reactor comprisesan elongate cavity, through which the first liquid composition and/orthe second liquid composition flow.

40. The method according to item 39, wherein the elongate cavity has theshape of a cylinder, a tube, a pipe, a capillary or a hose.

41. The method according to any one of items 37 to 40, wherein oneliquid composition selected from the first liquid composition and thesecond liquid composition flows through the reactor and the respectiveother liquid composition is injected into the liquid that flows throughthe reactor, wherein the respective other liquid composition is injectedin the reactor or upstream of the reactor.

42. The method according to any one of the preceding items, wherein themethod is conducted continuously or discontinuously.

43. The method according to any one of the preceding items, wherein thefirst liquid composition and/or the second liquid composition areintroduced into the at least one reactor with a flow rate of 0.1ml/minute ore more, preferably 50 ml/minute or more, more preferably 100ml/minute or more, more preferably 150 ml/minute or more, morepreferably 300 ml/minute or more, more preferably 800 ml or more, mostpreferably in a range of 100-800 ml/minute.

44. The method according to any one of the preceding items, wherein thefirst liquid composition and the second liquid composition aresuccessively introduced into the at least one reactor.

45. The method according to any one of items 1 to 43, wherein the firstliquid composition and the second liquid composition are concurrentlyintroduced into the at least one reactor, preferably under controlledconditions e.g. constant flow rates.

46. The method according to item 45, wherein the first liquidcomposition and the second liquid composition are introduced into the atleast one reactor with the same flow rate.

47. The method according to any one of the preceding items, furthercomprising a step (e) of isolating or concentrating the nanoparticlecomprising at least one RNA and at least one cationic or polycationiccompound from the product liquid composition comprising the nanoparticlecomprising at least one RNA and at least one cationic or polycationiccompound.

48. The method according to item 47, wherein step (e) comprises a dryingstep, preferably a drying step selected from the group consisting oflyophilisation, spray-drying, or freeze-spray drying.

49. The method according to any one of the preceding items, wherein thenanoparticle comprising at least one RNA and at least one cationic orpolycationic compound, or the product liquid composition comprising thenanoparticle comprising at least one RNA and at least one cationic orpolycationic compound are for use in a pharmaceutical composition.

50. A liquid composition comprising the nanoparticle comprising at leastone RNA and at least one cationic or polycationic compound, which areobtainable by the method according to any one of items 1 to 47.

51. A nanoparticle comprising at least one RNA and at least one cationicor polycationic compound, obtainable by the method according to items 47or 48.

52. Use of the method according to any one of items 1 to 49 in themanufacture and/or preparation of a medicament or a vaccine, preferablya medicament or a vaccine for use in the treatment or prophylaxis of adisorder or a disease.

53. The use according to item 52, wherein the disorder or the disease isselected from the group consisting of cancer or tumor diseases,infectious diseases, preferably viral, bacterial or protozoologicalinfectious diseases, autoimmune diseases, allergies or allergicdiseases, monogenetic diseases, i.e. (hereditary) diseases, or geneticdiseases in general, diseases which have a genetic inherited backgroundand which are typically caused by a single gene defect and are inheritedaccording to Mendel's laws, cardiovascular diseases and neuronaldiseases.

54. A pharmaceutical composition comprising the liquid compositioncomprising the nanoparticle comprising at least one RNA and at least onecationic or polycationic compound according to item 50, and/or thenanoparticle comprising at least one RNA and at least one cationic orpolycationic compound according to item 51.

55. The pharmaceutical composition according to item 54, wherein thepharmaceutical composition is for use in the treatment or prophylaxis ofa disorder or a disease.

56. The pharmaceutical composition for use according to item 55, whereinthe disorder or the disease is selected from cancer or tumor diseases,infectious diseases, preferably viral, bacterial or protozoologicalinfectious diseases, autoimmune diseases, allergies or allergicdiseases, monogenetic diseases, i.e. (hereditary) diseases, or geneticdiseases in general, diseases which have a genetic inherited backgroundand which are typically caused by a single gene defect and are inheritedaccording to Mendel's laws, cardiovascular diseases and neuronaldiseases.

BRIEF DESCRIPTION OF THE FIGURES

The figures shown in the following are merely illustrative and shalldescribe the present invention in a further way. These figures shall notbe construed to limit the present invention thereto.

FIG. 1 : G/C-enriched mRNA sequence R2564 coding for the hemagglutinin(HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)),corresponding to SEQ ID NO: 1.

FIG. 2 : Schematic flow diagram for a non-continuously operating reactorfor the preparation of RNA-comprising nanoparticles. (1) feed vessel;(2) pump; (3) open tank; (4) stirrer. The reference signs do only applyto that particular figure and are not used continuously; the referencesigns shall not be used, applied or translated to other figures anddrawings.

FIG. 3 : Schematic flow diagram for a continuously operating stirredreactor for the preparation of RNA-comprising nanoparticles. (1) Feedvessel (for example for the solution comprising the cationic orpolycationic compound); (2) Pump; (3) Feed vessel (for example for theRNA solution); (4) Pump; (5) continuously stirred tank; (6) Receivervessel for the RNA-comprising nanoparticles. This vessel may be replacedby further processing modules.

The reference signs do only apply to that particular figure and are notused continuously; the reference signs shall not be used, applied ortranslated to other figures and drawings.

FIG. 4 : Schematic view of a continuously operating stirred reactor(Reactor III, as prepared according to Example 3.3 described below). (1)Cylindrical reactor chamber; (2) Inlet port; (3) Inlet port; (4) Inletport; (5) Magnetic stirrer; (6) Stir bar. The reference signs do onlyapply to that particular figure and are not used continuously; thereference signs shall not be used, applied or translated to otherfigures and drawings.

FIG. 5 : Schematic view of a continuously operating stirred reactor(Reactor V, cf. Example 3.5 described below). (1) Stainless steelreactor body; (2) Inlet connection for silicone hose tube (addition ofthe RNA solution); (3) Inlet connection for silicone hose tube (additionof the cationic or polycationic compound solution, e.g. protaminesolution); (4) Reactor chamber; (5) Outlet connection for silicone hosetube, (6) Magnetic stirrer; (7) Stir bar; (8) Ventilation connection(for bubble-free filling of reactor, closed under operation); (9) Sealring. A technical drawing of that reactor type is provided in FIG. 13 .The reference signs do only apply to that particular figure and are notused continuously; the reference signs shall not be used, applied ortranslated to other figures and drawings.

FIG. 6 : Schematic view of a continuously operating reactor (Reactor VI,cf. Example 3.6 described below). (1) Reactor comprising static mixingelements; (2) Inlet connection for silicone hose tube (addition of theRNA solution); (3) Inlet connection for silicone hose tube (addition ofthe cationic or polycationic compound solution, e.g. protaminesolution); (4) Reactor chamber; (5) Staticmixing elements; (6) Outletconnection for silicone tube. Theeference signs do only apply to thatparticular figure and are not used continuously; the reference signsshall not be used, applied or translated to other figures and drawings.

FIG. 7 : Photograph of product dispersions of RNA-protaminenanoparticles in 96-well microtiter plates produced in Examples 4 to 8(cf. Example 2.2). Undesired precipitates are visible in some reactions(grey colour; indicated by asterisks). These precipitates were formed inexperiments where the blend time was 8 seconds or longer (cf. Table 5).

FIG. 8 : Summary of blend time of reactors (cf. Example 3), andclarity/turbidity, particle size and polydispersity of RNA-protaminenanoparticles produced in Examples 4 to 8 (cf. Example 2): Effect ofreactor design, stir rate and total flow rate on the blend time (toppanel, average of 5 measurements with standard deviation), theabsorption at 350 nm (A350, second panel), on particle size (Z-average,third panel) and the particle polydispersity (fourth panel).

FIG. 9 : Immunostimulation by RNA-comprising nanoparticles. Theinduction of cytokines in peripheral blood mononuclear cells (PBMCs) byRNA/protamine nanoparticles, which were produced in Examples 5.1 or 8.4,is shown (cf. Example 10).

FIG. 10 : Schematic view of a continuously operating reactor (ReactorVI, cf. Example 3.6 described below). (1) Reactor comprising staticmixing elements; (2) pumps for reagent solutions; (3) T-piece adapter.The reference signs do only apply to that particular figure and are notused continuously; the reference signs shall not be used, applied ortranslated to other figures and drawings.

FIG. 11 : Schematic view of a continuously operating reactor (ReactorVII, cf. Example 3.7 described below). (1) Reactor comprisinginjector-type mixing means; (2) Inlet connection for silicone tube(addition of the RNA solution); (3) Inlet connection for silicone tube(addition of the cationic or polycationic compound solution, e.g.protamine solution); (4) Reaction zone; (5) injection angle α; (6)Outlet connection for silicone tube. The reference signs are inbrackets. The reference signs do only apply to that particular figureand are not used continuously; the reference signs shall not be used,applied or translated to other figures and drawings. Other indicatednumbers represent distances in mm.

FIG. 12 : Schematic view of the continuously operating injector mixer(Reactor VII) used in Example 3.7. The injector mixer mixer consisted ofa stainless steel housing with an inner diameter of 3.4 mm and a lengthof 30 mm. Inserted was a Sulzer SMX static mixer DN3 with a length of 22mm and a diameter of 3.2 mm made of stainless steel.

FIG. 13 : Technical drawing of a schematic view of a continuouslyoperating stirred reactor (Reactor V, cf. FIG. 5 and Example 3.5 asdescribed below). Indicated numbers represent distances in mm and do notrefer to reference signs.

FIG. 14 : Schematic simplified reactor models used for computationalfluid dynamics (CFD) analysis. In panel (A) a model of reactor type V(continuous reactor with stirring means) is shown and in panel (B) areactor type V.1 (continuous reactor without stirring means) is shown(see Example 3). Reference signs: (1) inlet port (addition of RNAcontaining solution); (2) inlet port (addition of the cationic orpolycationic compound solution); (3) stirring means (magnetic stirrer);(4) outlet port. Further details of the CFD analysis are provided inExample 12. The reference signs do only apply to that particular figureand are not used continuously; the reference signs shall not be used andapplied or translated to other figures and drawings.

FIG. 15 : Exemplary result of a CFD analysis, performed according toExample 12. The graph shows the homogeneity/uniformity obtained at theoutlet port after a certain time period. The dashed line (asterisk)indicates the blend time of the mixing process (reactor V, 160 ml/min,1400 rpm) required in order to reach a given homogeneity, in the contextof the invention, to reach a homogeneity level of 0.8 or 80%. Furtherdetails of the CFD analysis are provided in Example 12.

FIG. 16 : Exemplary result of a CFD analysis, performed according toExample 12. The graph shows the homogeneity/uniformity obtained at theoutlet port after a certain time period. The figure shows that asufficient homogeneity of the mixture could not be obtained in thesimulated time interval for reactor V.1 at 10 ml/min flow rate. In thecontext of the invention, a homogeneity level of 0.8 or 80% is desired,indicated by a horizontal line. Therefore, a blend time could not bedetermined. Further details of the CFD analysis are provided in Example12.

EXAMPLES

The Examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These Examples shallnot be construed to limit the present invention thereto.

In the following examples according to the present invention andcomparative examples, the method according to the present invention iscarried out in some exemplary reactors, which are described in detail inExample 3 using standardised model reagent compositions of RNA andcationic or polycationic compound, respectively, which are described indetail in Example 1, and the respective products are characterisedaccording methods, which are described in detail in Example 2.

Example 1—Reagents Used in all Examples and Reference Examples

1.1 Preparation of the RNA Solution

1.1.1 Preparation of DNA and mRNA Constructs

For the model reaction used most Examples and Reference Examplesdescribed in the following, a DNA sequence encoding the hemagglutinin(HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)) wasprepared and used for subsequent in vitro transcription reactions.

In this embodiment, the DNA sequence encoding the above-mentioned mRNAwas prepared.

The construct R2564 (Influenza HA encoding mRNA: SEQ ID NO: 1, shown inFIG. 1 ) was prepared by introducing a 5′-TOP-UTR derived from theribosomal protein 32L4, modifying the wild type coding sequence byintroducing a GC-optimized sequence for stabilization, followed by astabilizing sequence derived from the albumin-3′-UTR, a stretch of 64adenosines (poly(A)-sequence), a stretch of 30 cytosines(poly(C)-sequence), and a histone stem loop. The sequence (SEQ ID NO: 1)of the corresponding mRNA is shown in FIG. 1 .

1.1.2. In Vitro Transcription of RNA

The respective DNA plasmid prepared according to section 1.1 above wastranscribed in vitro using T7 RNA polymerase (Thermo Fisher ScientificInc.). The in vitro transcription of influenza HA encoding R2564 wasperformed in the presence of a CAP analog (m⁷GpppG). Subsequently theRNA was purified using PureMessenger® (CureVac, Tubingen, Germany;WO2008/077592A1).

1.1.3 Preparation of Standard RNA Solution

A standard RNA solution was prepared in purified water wherein theconcentration of RNA was 0.87 g/L, further comprising about 9 mM Na andabout 6.5 mM Cl. The ratio Na/RNA was about 10.3 mmol/g. The pH of thesolution was 5.8.

In other experiments, a standard RNA solution was prepared in purifiedwater, wherein the concentration of RNA was 0.87 g/L. The pH of thesolution was 5.8.

1.2 Preparation of the Protamine Solution

For the model reaction used in all Examples and Reference Examplesdescribed in the following, a standard solution containing protamine(Meda Pharma) and trehalose (Ferro Pfanstiehl) was prepared in purifiedwater. The concentration of protamine was 0.43 g/L (corresponding to43.9 Units/mL), and the concentration of trehalose was 10.87% (w/w). Forrespective model reactions using that protamine solution comprisingprotamine obtained from Meda Pharma, the RNA solution did compriseadditional NaCl (cf. 1.1.3).

For other Examples, GMP-grade protamine (LeoPharma GmbH; comprising 147mM NaCl) was used. The concentration of GMP-grade protamine was 0.314g/L (correspornding to 43.9 Units/mL of protamine), and theconcentration of trehalose was 10.87% (w/w). For respective modelreactions using the protamine solution comprising GMP-grade protamine,the RNA solution did not comprise additional NaCl (cf. 1.1.3).

Example 2—Characterisation of Product Dispersions

2.1 Characterization of Nanoparticles

Measurement of Particle Size and Polydispersity

The hydrodynamic diameter of the nanoparticles was measured by dynamiclight scattering using a Zetasizer Nano ZS (Malvern Instruments,Malvern, UK) according to the instructions provided by the manufacturer.The measurements were performed at 25° C. and a scattering angle of 173°in the specified buffer analysed by a cumulant method to obtain thehydrodynamic diameters and polydispersity indices of the nanoparticles.

For measuring the particle size of the RNA-protamine nanoparticlesobtained in dispersion according to the method of the present inventionin the Examples and Reference Examples, 70 μl of the respective productsolution was filled into a UV transmittable cuvette (UVette, Eppendorf),which in turn was placed into a Zetasizer nano ZS (Malvern instruments)and the measurement was conducted using the following settings:Refractive index of material: 1.450;

absorption of material: 0.001; dispersant temperature: 25° C.;dispersant viscosity: 0.8753; dispersant refractive index: 1.331;Mark-Houwink parameters: A parameter 0.428; K-parameter 7.67⁻⁰⁵ cm²/s;use dispersant viscosity as sample viscosity; sample temperature: 25°C.; sample cuvette: Zen 0040 disposable cuvette, equilibration time: 0s; measurement angle: 173° backscatter; automatic measurement duration;number of measurements: 1; automatic attenuation setting; positioningmethod: seek for optimal position; no extension duration for largeparticles; analysis model: normal resolution. The average particle size(hydrodynamic diameter of a particle in nm) is given as the Z-averageand the polydispersity is given as the polydispersity index (PDI), bothof which were calculated by the instrument's software (Zetasizersoftware version 6.34, Malvern Instruments).

2.2 Characterization of Product Solution

2.2.1 Measurement of the Absorption at 350 nm (A350)

The transmitted light through a sample solution can be measured in aUV-Vis spectrophotometer at a wavelength where the ingredients, such asproteins, peptides, DNA/RNA, and formulation excipients do not absorblight, i.e., typically in the range of 320-800 nm.

To rapidly measure and compare the clarity of the product nanoparticledispersion (dispersion comprising the RNA-protamine nanoparticles), theabsorption at 350 nm (A350) was determined. 200 μl of each productdispersion were applied to a microwell plate (Costar, UV Plate, 96 well,no lid, UV transmittable flat bottom). A350 was measured with a SynergyHT plate reader (BioTek systems). Pathlength correction was performed byGen5 software (BioTek, Installation version: 1.11.5) with a testwavelength of 977 nm and a reference wavelength of 900 nm. Correctionwas performed with a constant K-factor of 0.18 to yield the A350 valuecorrected to 1 cm pathlength.

2.2.2 Photographic Documentation

Images of the plates from Example 2.2.1 were taken with an E-box VX2(Vilber) documentation system.

2.2.3 Measurement of Turbidity (FNU)

The turbidity of the product nanoparticle dispersions was measured witha NEPHLA turbidimeter (Dr. Lange, Düsseldorf, Germany), operating at 860nm and detecting at 90° angle. The system is calibrated with formazin asstandard and the results were given in formazin nephelometric units(FNU). For the measurement, 2.0 ml of the formulations were analyzed.

Example 3—Characterization of Reactors

In the Examples and Reference Examples, the following reactors were usedas exemplary embodiments of chemical reactors of different generaldesign.

3.1 Reactor I—Non-Continuous Reactor (Non-Continuous Dynamic Mixer)

A well of a deep well plate (MegaBlock® 96 Well, 2.2 ml, polypropylenefrom Sarstedt) mounted on a shaking device (Multi-MicroPlate Genie fromScientific Industries) was used as an example of an open micro-reactor(Reactor I). Addition of the reagent solutions and removal of theproduct dispersion after reaction were carried out successively by meansof a pipette.

3.2 Reactor II—Non-Continuous Reactor with Stirring Means(Non-Continuous Dynamic Mixer)

A reactor (Tube 30 ml, 95×24.8 mm, polypropylene from Sarstedt,)equipped with a 1.7×0.4 mm stirring bar (VWR) mounted on a magneticstirrer (500 or 1000 rpm, Thermo Compact 20, available from ThermoFisher Scientific Inc) was used as an example of an open batchmicro-reactor with stirring (Reactor II) The reactor design isschematically shown in FIG. 2 , 3.15 mg of RNA (R2564) in a total volumeof 605 μl resulting in an RNA concentration of 5.2 g/L were provided inthe reactor. 3.66 ml of a protamine/trehalose mixture (0.235 g/Lprotamine, 5.92% w/w trehalose) were added with a syringe pump (TSEsystems 540060-B) and an addition speed (flow rate) of 0.37 ml/min.

3.3 Reactor III—Continuous Reactor with Stirring Means/Design 1(Continuous Dynamic Mixer)

An example of a continuously stirred micro-reactor (Reactor III) havinga cylindrical reactor chamber with a volume of 400 μl (diameter: 12.5mm; hight: 3.3 mm) was constructed from a 5 ml syringe with Luer lock(Braun Melsungen; Germany) and equipped with a cylindrical stirring bar(8×1.5 mm, VWR, Germany). The reactor design is schematically shown inFIG. 4 , wherein the reactor body is indicated by reference sign (1) ofFIG. 4 , and the stirring bar is indicated by reference sign (6) of FIG.4 . Two inlet ports (reference signs 3, 4 of FIG. 4 ) for introducingthe two reagent solutions were provided at opposite sides of the reactorchamber by means of butterfly needles pierced into the side wall of thechamber just below the top cover of the chamber, and an outlet port(reference sign 2 of FIG. 4 ) for recovering the product dispersion wasprovided by another butterfly needle attached to the Luer lockpositioned in the top cover of the chamber. The chamber was mounted on amagnetic stirrer (reference sign (5) of FIG. 4 , 200 or 1000 rpm, ThermoCompact 20, available from Thermo Fisher Scientific Inc). Duringoperation, the additions of the reagent solutions were carried out bymeans of syringe pumps (TSE systems 540060-Bnot shown in FIG. 4 ) eachwith flow rates of 0.1 to 0.5 ml/min. (addition speed), while theremoval of the product dispersion after reaction was achieved by thecombined flow provided thereby.

3.4 Reactor IV—Continuous Reactor with Stirring Means/Design 2(Continuous Dynamic Mixer)

An example of a continuously stirred micro-reactor (Reactor IV) having acylindrical reactor chamber with a volume of 1500 μl (diameter: 15.89mm; hight: 7.6 mm) was constructed from a 10 ml syringe with Luer lock(Braun Melsungen; Germany) and equipped with an oval stir bar (15×6mmVWR, Germany). Two inlet ports for introducing the two reagentsolutions were provided at opposite sides of the reactor chamber bymeans of butterfly needles pierced into the side wall of the chamberjust below the top cover of the chamber, and an outlet port forrecovering the product dispersion was provided by another butterflyneedle attached to the Luer lock positioned at the bottom of thechamber. The chamber was mounted on a magnetic stirrer (200 or 1400 rpm,Thermo Compact 20, available from Thermo Fisher Scientific Inc). Duringoperation, the addition of the reagent solutions were carried out bymeans of syringe pumps (TSE systems 540060-Beach with flow rates of 0.1to 5 ml/min. (addition speed), while the removal of the productdispersion after reaction was achieved by the combined flow providedthereby.

3.5.1 Reactor V—Continuous Reactor with Stirring Means/Design 3(Continuous Dynamic Mixer)

An example of a continuously stirred micro-reactor (Reactor V) having acylindrical reactor chamber with a volume of 2.1 ml (diameter: 26 mm;hight: 4 mm) was constructed from stainless steel. The reactor design isschematically shown in FIG. 5 , wherein the reactor body is indicated byreference sign (1, FIG. 5 ) and the cylindrical reaction chamber isindicated by reference sign (4, FIG. 5 ). The reactor comprises twoinlet ports (reference signs 2, 3 of FIG. 5 ) for introducing the tworeagent solutions, which are located at opposite sides of the reactorchamber, and an outlet port (reference sign 5 of FIG. 5 ) for recoveringthe product dispersion, which is located in the centre of the bottom ofthe chamber. Further, the reactor chamber was equipped with acylindrical stir bar (Reference sign (7, FIG. 5 ), 8×3 mm, VWR,Germany), and the reactor chamber was mounted on a magnetic stirrer(reference sign (6, of FIG. 5 ), 200 or 1000 rpm, Thermo Compact 20,available from Thermo Fisher Scientific Inc.). The lid of the reactorchamber was tightly sealed with a seal ring (reference sign 9 of FIG. 5) made from polytetrafluoroethylene (PTFE) and provided with aventilation means (reference sign 8 of FIG. 5 ) to allow for bubble-freefilling of the reactor chamber, which was closed after complete fillingof the reactor chamber. During operation, the addition of the reagentsolutions were carried out by means of syringe pumps (TSE systems540060-Bnot shown in FIG. 5 ) with flow rates of 0.1 to 0.5 ml/min.(addition speed), while the removal of the product dispersion afterreaction was achieved by the combined flow provided thereby. A technicaldrawing of the reactor type V is provided in FIG. 13 .

3.5.2 Reactor V.1—Continuous Reactor without Stirring Means/Design 3.2(Continuous Static Mixer)

An adapted version of the continuous dynamic mixer according to 3.5.1.In some experiments, the reactor was used without a stir bar. Atechnical drawing of the reactor type V is provided in FIG. 13 .

3.6 Reactor VI—Reactor with Static Mixing Means (Static Mixer)

An example of a reactor comprising a static mixing means (Reactor VI)was constructed. The static mixer consisted of a stainless steel housingwith an inner diameter of 3.4 mm and a length of 30 mm. Inserted was aSulzer SMX static mixer DN3 with a length of 22 mm and a diameter of 3.2mm made of stainless steel.

The reactor principle is schematically shown in FIG. 6 . A more detaileddrawing is shown in FIG. 12 . One opening of the tube is provided withtwo inlet ports for introducing the reagent solutions with pumps (notshown) at different flow rates. The reactor was connected to a neMESYSsyringe pump (Cetoni) used for the introducing and mixing of 0.87 mg/mlRNA and protamine-trehalose solution (10.86% trehalose, 0.43 mg/mlprotamine) in a plastic T-piece adapter and a stainless steel staticmixing device from Sulzer (see above) (FIG. 6 , FIG. 10 and FIG. 12 ).Both solutions were pumped through the static mixer with various totalflow rates. The product dispersion is obtained after a reaction way of22 mm.

3.7 Reactor VII—Reactor with Injector System as Mixing Means (StaticMixer)

An example of a reactor comprising a mixing means in form of an injectorsystem (Reactor VII) was constructed by providing an inlet port at anangle a of 30° at the side wall of a tube reactor as shown in FIG. 11 .[41.5 mm length, 6-3 mm reactor diameter, angle about 30°, diameterinjection tube 0.5 mm) The two inlet ports are provided with a neMESYSsyringe pump (Cetoni) (not shown) to provide the reagent solutions withdifferent flow rates. The product dispersion is obtained after areaction way of 41.5 mm.

3.8 Measurement of the Blend Time

The blend time was measured for each of the reactors I to V according tothe following procedure. The reaction chamber was filled completely witha solution of 75% Glycerol/0.01 M NaOH/0.01% Bromophenol blue in water(solution 1). Under stirring (using a cylindrical stirring bar, (8×3 mm,VWR, Germany) and a magnetic stirrer (Thermo Compact 20, available fromThermo Fisher Scientific Inc.) (but without flow-through for reactorsIII to V), a volume of 0.01 M HCl corresponding to 0.01 volumeequivalents of solution 1 were added and the time until the colour haschanged from purple to yellow was recorded visually by a stop watch inquintuplicates.

The averaged blend rates measured for reactors I to V are summarised inTable 5.

In addition to the above described experimental procedure, the blendtimes of reactor types V and V.1 were determined using computationalfluid dynamics (CFD) analysis for different conditions (e.g. differentflow rates) (see Example 12).

Example 4: Preparation of RNA-Comprising Nanoparticles in Reactor I

130.5 μg of RNA (obtained in Example 1.1.2) in 30 μl water for injectionwere added to Reactor I (cf. Example 3.1). 270 μl of aprotamine/trehalose solution (0.242 g/L protamine, 6.025% w/w trehalosein water for injection) were added stepwise by pipetting over 10 minutes(10 steps a 27 μl with dispensing and a 1 minute interval between eachstep) under shaking. After completion of the addition, the productdispersion was completely removed from the reactor by pipette and theproduct dispersion was analysed according to Example 2. The results aresummarised in Table 5 and shown in FIGS. 7 and 8 as Experiment I.

Example 5: Preparation of RNA-Comprising Nanoparticles in Reactor II

3.15 mg of RNA (R2564, obtained in Example 1.1.2) in a total volume of605 μl resulting in an RNA concentration of 5.2 g/L were added toReactor II. 3.66 ml of a protamine/trehalose mixture (0.235 g/Lprotamine, 5.92% w/w trehalose) were added with a syringe pump and anaddition speed (flow rate) of 0.37 ml/min. Mixing in the reactor wasperformed with a stir bar (500 or 1000 rpm). The reaction conditions aresummarised in Table 1). After completion of the addition, the productdispersion was completely removed from the reactor by pipette and theproduct dispersion was analysed according to Example 2. The results aresummarised in Table 5 and shown in FIGS. 7 and 8 .

TABLE 1 Stir and addition rates for Reactor II Addition rate ofprotamine/ Total Magnetic Experiment trehalose solution flow ratestirrer setting number (ml/min) (ml/min) (rpm) II.A 0.37 0.37 500 II.B0.37 0.37 1000

Example 6: Preparation of RNA-Comprising Nanoparticles in Reactor III

The standard RNA solution (0.87 g/L) prepared in Example 1.1.3 and theprotamine/trehalose standard solution (0.43 g/L protamine; 10.87%trehalose) prepared in Example 1.2 were pumped into Reactor III with theflow and stir rates summarised in Table 2. The results are summarised inTable 5 and shown in FIGS. 7 and 8 .

TABLE 2 Stir and addition rates for Reactor III Addition rate Additionrate of protamine/ of RNA trehalose Total Magnetic Experiment solutionsolution flow rate stirrer setting number (ml/min) (ml/min) (ml/min)(rpm) III.A 0.1 0.1 0.2 200 III.B 0.5 0.5 1 200 III.C 0.1 0.1 0.2 1000III.D 0.5 0.5 1 1000

Example 7: Preparation of RNA-Comprising Nanoparticles in Reactor IV

The standard RNA solution (0.87 g/L) prepared in Example 1.1.3 and theprotamine/trehalose standard solution (0.43 g/L protamine; 10.87%trehalose) prepared in Example 1.2 were pumped into Reactor IV with theflow and stir rates summarised in Table 3. The results are summarised inTable 5 and shown in FIGS. 7 and 8 .

TABLE 3 Stir and addition rates for Reactor IV Addition rate Additionrate of protamine/ of RNA trehalose Total Magnetic Experiment solutionsolution flow rate stirrer setting number (ml/min) (ml/min) (ml/min)(U/min) IV.A 0.1 0.1 0.2 200 IV.B 0.1 0.1 0.2 1000 IV.C 5.5 5.5 11 1000

Example 8: Preparation of RNA-Comprising Nanoparticles in Reactor V

The standard RNA solution (0.87 g/L) prepared in Example 1.1.3 and theprotamine/trehalose standard solution (0.43 g/L protamine; 10.87%trehalose) prepared in Example 1.2 were pumped into Reactor V with theflow and stir rates summarised in Table 4. The results are summarised inTable 5 and shown in FIGS. 7 and 8 .

TABLE 4 Stir and addition rates for Reactor V Addition rate Additionrate of protamine/ of RNA trehalose Total Magnetic Experiment solutionsolution flow rate stirrer setting number (ml/min) (ml/min) (ml/min)(U/min) V.A 0.5 0.5 1 200 V.B 0.1 0.1 0.2 1400 V.C 0.5 0.5 1 1400 V.D1.5 1.5 3 1400 V.E 5 5 10 1400 V.F 1.5 1.5 3 1400 V.G 50 50 100 1400 V.H50 50 100 1400 V.I 50 50 100 1400

Results

The results of the experiments described in Examples 4 to 8 aresummarised in Table 5 and shown in FIGS. 7 and 8 .

TABLE 5 Results of Examples 4 to 9 A350 (pathlength Z-average ExperimentBlend time corrected (diameter Polydispersity number [s] to 1 cm) [nm])index I* 48 0.716 176 0.133 II.A* 4 0.242 115 0.131 II.B* 3 0.2 99 0.137III.A 41 0.961 172 0.147 III.B 41 1.1 183 0.153 III.C 8 0.635 149 0.126III.D 8 0.562 143 0.097 IV.A 17 0.781 155 0.164 IV.B 2.2 0.373 116 0.149IV.C 2.2 0.485 130 0.109 V.A 13 0.526 148 0.160 V.B 2 0.331 120 0.141V.C 2 0.346 135 0.179 V.D 2 0.295 115 0.096 V.E 2 0.323 121 0.139 V.F>0.1^($) 0.44 135 0.14 V.G >0.1^($) 0.24 126 0.09 V.H >0.1^($) 0.26 1160.11 V.I >0.1^($) 0.27 112 0.14 *reaction performed non-continuously^($)approximation

From the data summarised in Table 5 and shown in FIGS. 7 and 8 , thefollowing may be deduced.

Using a pipette and microplate shaker for mixing without stirring thesolution (method I, Example 4) resulted in samples of low clarity/highturbidity (FIG. 7 ) with A350 values above 0.5 and in largeRNA/protamine nanoparticles/complexes with a Z-average above 150 nm(Table 5 and FIG. 8 ).

In contrast, when protamine was added to an RNA solution at a constantflow rate and under stirring according to the method of the presentinvention (Method II, Example 5), the product dispersion wassignificantly clearer than with Method I (FIG. 7 ) with A350 valueswell-below 0.5 and the particles sizes were decreased to sizes of about120 nm (Table 5 and FIG. 8 ).

When using one of the five configurations of dynamic mixers (stirredreactors; Reactors I, II, III, IV and V), the product quality iscontrolled by the blend time.

Reactor III (Example 4) exhibits a blend time of more than 40 s when thestirring means is operated at 200 rpm, and a blend time of 8 s when thestirring means is operated at 1000 rpm. In both cases, the productdispersions were turbid with precipitating material observed at thelowest mixing efficiency (blend time >40 s and 1 ml/min total flowrate). Clear dispersions and A350 values below 0.5 could not be achievedunder these conditions. In contrast, Reactor IV (Example 5) exhibitslower blend times, namely a blend time of 17 s when the stirring meansis operated at 200 rpm, and a blend time of 2 s when the stirring meansis operated at 1000 rpm. Using this reactor in the method according tothe present invention, clear product dispersions having an A350 of lessthan 0.5 are obtained, independent of the total flow rates of 0.2 ml/mland 11 ml/min but dependent on the stirring rate.

For scaling-up the production process, Reactor V exhibiting furtherdecreased blend times (Example 6), namely a blend time of 13 s when thestirring means is operated at 200 rpm, and a blend time of 2 s when thestirring means is operated at 1400 rpm, was used in the method accordingto the present invention. Thus, clear product dispersions having an A350of less than 0.5 and particle sizes of about 120 nm were obtained withflow rates from 0.2 to 10 ml/min, when the stir rate was 1400 rpm.However, with a stir rate of 200 rpm, the product dispersion was lessclear with an A350 value of about 0.5.

In addition, higher flow rates were tested (V.F−V.I). Those experimentsclearly show that a higher flow rate resulted in dispersions with lessprecipitates and also smaller and more uniform nanoparticle sizes. Theblend time in this setup (100 ml/min flow rate, 1400 rpm stirring) wasestimated to be shorter than 0.1 seconds.

To determine the blend time in setups with higher flow rates, CFDanalysis for 50 ml/min and 160 ml/min flow rates were conducted (seeExample 12).

It has to be noted that only for blend times shorter than 5 seconds,nanoparticles containing products could be generated while avoiding theformation of precipitates (A350 smaller than 0.5; cf. FIG. 7 ; reactionswith precipitates are indicated with an asterisk). In addition, it hasbeen surprisingly found that increasing the blend time decreased theprecipitation and therefore the quality of the product solution.

In summary, the above examples demonstrate that only the productdispersions obtained by the method according to the present inventionexhibit the properties required for use of the RNA in the medical field,as well as the required production reproducibility.

Example 9: Preparation of RNA-Comprising Nanoparticles in Reactor VI

The standard RNA solution (0.87 g/L) prepared in Example 1.1.3 and theprotamine/trehalose standard solution (0.43 g/L protamine; 10.87%trehalose) prepared in Example 1.2 were pumped into Reactor VI with thetotal flow rates, which are summarised in Table 6 together with theaverage particle sizes (zetasizer, Z-avarages), polydispersity index(PDI), absorption at 350 nm (A350) and turbidity, determined accordingto Example 2.

TABLE 6 Results and parameters for Reactor VI total Z-avarage Experimentflow rate (diameter Turbidity number [μl/s] [nm]) PDI A350 [FNU] VI.A240 86.1 0.259 0.097 7.8 VI.B 300 186.6 0.239 0.258 39.0 VI.C 500 161.90.247 0.235 35.5

Results: In all performed experiments, it could be shown that particlessmaller than 200 nm in average were generated in clear dispersions,without causing increased precipitation (A350<0.5). Lower flow ratesseem to correlate with smaller particle sizes and less precipitation(low A350 nm values).

Example 10: Preparation of RNA-Comprising Nanoparticles in Reactor VII

The standard RNA solution (0.87 g/L) prepared in Example 1.1.3 and theprotamine/trehalose standard solution (0.43 g/L protamine; 10.87%trehalose) prepared in Example 1.2 were pumped into Reactor VII with thetotal flow rates summarised in Table 7 together with the averageparticle sizes (zetasizer, Z-avarages), polydispersity index (PDI),absorption at 350 nm (A350) and turbidity, determined according toExample 2. Experiments with flow rates of 400 μl/s and 550 μl/s wererepeated.

TABLE 7 Results and parameters for Reactor VII total Z-avarageExperiment flow rate (diameter (A350) Turbidity number [μl/s] [nm]) PDIOD 350 nm [FNU] VII.A 200 253.8 0.338 0.729 161.4 VII.B 275 153.0 0.1760.533 101.5 VII.C 400 145.0 0.188 0.437 79.4 VII.C-2* 400 166.3 0.2310.389 72.9 VII.D 440 96.6 0.180 0.172 17.2 VII.E 550 90.9 0.180 0.13010.0 VII.E-2* 550 78.6 0.153 0.101 6.1 VII.F 600 70.4 0.153 0.089 4.6*experiment repeated

Results: In the performed injector mixing experiments, it could be shownthat particles larger than 200 nm in average were generated with flowrates of 200 μl/s. The absorption at 350 nm (A350) moreover indicatesthat also precipitation occurred using such low flow rates. Both, theaverage particle size, and the A350 values decreased by increasing flowrates. With flow rates between 400 and 600 μl/s, on average a particlesize smaller than 200 nm and an A350 value below 0.5 could be reached,and therefore define a preferred range.

In summary, the above examples demonstrate that only the productdispersions obtained by the method according to the present inventionexhibit the properties required for RNA therapeutics, as well as therequired production reproducibility.

Example 11: Stimulation of Cytokines in Peripheral Blood MononuclearCells

In this test of immunostimulation, the dispersions comprising RNA(R2564)-protamine nanoparticles obtained in Experiments II.A and V.Dwere used. For a formulation with free RNA, the dispersions weresupplemented with R2564 to yield final concentrations of 0.4 g/L RNAcomplexed with 0.2 g/L protamine and 0.4 g/L free RNA.

The samples were lyophilized (Christ Alpha I) and reconstituted inRinger lactate (in the volume of the sample before lyophilization).Peripheral blood mononuclear cells (PBMCs) from healthy human subjectswere isolated by density gradient centrifugation and aliquots werecryopreserved in liquid nitrogen. On the day of the stimulation, PBMCswere thawed and 2×10⁵ cells seeded in each well of a 96 flat bottomplate in 200 μl X-Vivo 15 serum-free medium supplemented with 100 lUm/mlpenicillin/streptomycin (both Lonza). Cells were stimulated withR2564/II.A and R2564/V.D (concentration range: 114 to 14.2 nM).Untreated cells were used as control. After 24 hours, cell-freesupernatants were collected and the concentrations of TNF, IFN-α andIL-12p70 were measured by Cytometric Bead Array (CBA) according tomanufacturer's instructions (BD Biosciences; cf. Table 8) using the kitsof Table 8. Samples were acquired on a BD FACS Canto™ (BD Biosciences)and the data was analyzed using the FCAP Array v3.0 software (BDBiosciences). Both formulations showed comparable immunostimulation incell assays (FIG. 9 ).

TABLE 8 Kits from BD Biosciences used for measurement of cytokines incell culture supernatants. Reagent Catalog number Human Soluble ProteinMaster Buffer Kit 558264 Assay Diluent 560104 Human IFNα Flex Set 560379Human IL-12p70 Flex Set 558283 Human TNF Flex Set 560112

Results:

RNA-comprising nanoparticles produced by both methods according to themethod of the present invention were shown to induce comparable cytokinelevels in hPBMCs.

In summary, it is demonstrated that the method according to the presentinvention produces nanoparticles, which are characterised by havinguniform average particles sizes and polydispersity, reliably undercontrolled conditions, without allowing unwanted side reactionsresulting in unwanted side products (e.g. precipitates) and stabilityproblems caused thereby, independent of the scale of production.Further, the method according to the present invention is bothcost-effective and reliable, even on a large scale, which renders themethod of the invention especially suitable for the pharmaceuticalproduction of RNA-comprising nanoparticles on an industrial level.

Example 12: CFD Simulations of Reactor Types V and V.1

A computational fluid dynamics (CFD) analysis was performed in order todetermine the influence of flow-rate and mixing reactor geometry on theblending of the RNA solution and the protamine-trehalose solution. CFDanalysis was performed using a Star CCM+ software package.

In the computational model, the physical characteristics of the RNAsolution were assigned with a density of 997.9 kg/m³ and a kinematicviscosity of 2.39 mm²/s; the physical characteristics of theprotamine-trehalose solution were assigned with a density of 1039.4kg/m³ and a kinematic viscosity of 1.206 mm²/s.

The CFD analysis was performed for models of reactor type V (continuousreactor with stirring means) and reactor type V.1 (continuous reactorwithout stirring means) (see FIG. 14 and FIG. 16 ).

The blend time at different flow rates for certain reactor models wasdetermined according to the volume fraction of the protamine/trehalosesolution at the outlet port. Using this method, the homogeneity of theproduct liquid composition was simulated over time (an exemplary resultis shown in FIG. 15 ). The blend time was determined as the time point,where the mixture of both solutions showed a homogeneity/uniformity ofat least 80% (or 0.8).

Results:

In the CFD analysis, different parameters such as speed distribution atdifferent flow rates and medium distribution in the reactor models wereanalysed. The parameters included into the model, as well as theobtained results are summarized in Table 9. FIG. 15 and FIG. 16 shows anexemplary result of how the blend time has been determined.

TABLE 9 Results of Examples 12 Experiment Reactor Blend time Flow rateStirrer number type [s] [ml/min] (U/min) 1 V <1 50 1400 2 V <0.04 1601400 3 V.1 ## 10 4 V.1 <0.04 160 ##: A homogeneity level of 80% couldnot be achieved; therefore the blend time could not be determined (seeFIG. 16).

In summary, the results of the CFD simulation show that a fast andstable formulation of the first and the second liquid composition can beobtained with both reactor types at high flow rates (correlating toshort blend times). Surprisingly it has been found that a blend timeshorter than 1 second (at a flow rate of 50 ml/min) or a blend timeshorter than 0.04 seconds (at flow rates of 160 ml/min) was sufficientto obtain a homogeneous and uniform mixture.

The invention claimed is:
 1. A method for producing a liquid compositioncomprising a nanoparticle comprising at least one long-chain RNA and atleast one cationic or polycationic compound, wherein the methodcomprises the steps of: (a) providing a first liquid compositioncomprising the at least one long-chain RNA that comprises from 200 to15,000 nucleotides, (b) providing a second liquid composition comprisingthe at least one cationic or polycationic compound, (c) introducing thefirst liquid composition and the second liquid composition into at leastone reactor, wherein the first liquid composition and/or the secondliquid composition are introduced at a flow rate of at least 5mL/minute, and (d) recovering the product liquid composition comprisingthe nanoparticles comprising the at least one long-chain RNA and the atleast one cationic or polycationic compound from the reactor.
 2. Themethod according to claim 1, wherein the product liquid compositioncomprising the nanoparticle comprising the at least one long-chain RNAand the at least one cationic or polycationic compound comprises RNA inan amount of at least 1 g.
 3. The method according to claim 1, whereinthe at least one long-chain RNA comprises from 300 to 10,000nucleotides.
 4. The method according to claim 3, wherein the at leastone long-chain RNA is an mRNA.
 5. The method according to claim 1,wherein the nanoparticles comprising the at least one long-chain RNA andthe at least one cationic or polycationic compound have a particle sizeof 300 nm or less.
 6. The method according to claim 5, wherein thereactor comprises at least one dynamic mixing means.
 7. The methodaccording to claim 5, wherein the reactor comprises at least one staticmixing means.
 8. The method according to claim 5, wherein the reactorcomprises at least one T-piece adapter.
 9. The method according to claim8, wherein the nanoparticle comprising the at least one long-chain RNAand the at least one cationic or polycationic compound has apolydispersity index in a range from 0.05 to 0.50.
 10. The methodaccording to claim 9, wherein the first liquid composition and thesecond liquid composition are mixed with a blend time of 5 seconds orless.
 11. The method according to claim 8, wherein the at least onecationic or polycationic compound is selected from the group consistingof a cationic or polycationic peptide, a cationic or polycationicprotein, a cationic or polycationic polyamino acid, a cationic orpolycationic carbohydrate, a cationic or polycationic synthetic polymer,a cationic or polycationic small synthetic organic molecule, aninorganic multivalent cation, a cationic or polycationic lipid, acationic or polycationic polyamine compound, and a cationic orpolycationic polyimine compound.
 12. The method of claim 11, wherein theat least one cationic or polycationic compound is a cationic orpolycationic polyamino acid.
 13. The method of claim 11, wherein the atleast one cationic or polycationic compound is a cationic orpolycationic carbohydrate.
 14. The method of claim 11, wherein the atleast one cationic or polycationic compound is a cationic orpolycationic synthetic polymer.
 15. The method of claim 11, wherein theat least one cationic or polycationic compound is a cationic orpolycationic lipid.
 16. The method of claim 11, wherein the at least onecationic or polycationic compound is a cationic or polycationicpolyamine compound.
 17. The method according to claim 5, wherein thefirst liquid composition and the second liquid composition are added tothe at least one reactor so that the at least one cationic orpolycationic compound and the at least one long-chain RNA are present inthe reactor with an N/P-ratio in a range from 0.1 to
 10. 18. The methodaccording to claim 17, wherein the method is conducted continuously. 19.The method according to claim 17, wherein the first liquid compositionand/or the second liquid composition are introduced into the at leastone reactor with a flow rate of 10 mL/minute or more.
 20. The methodaccording to claim 19, wherein the first liquid composition and/or thesecond liquid composition are introduced into the at least one reactorvia a pump device.
 21. The method according to claim 20, wherein thefirst liquid composition and the second liquid composition areconcurrently introduced into the at least one reactor.
 22. The methodaccording to claim 21, further comprising a step (e) of isolating orconcentrating the nanoparticle comprising the at least one long-chainRNA and the at least one cationic or polycationic compound from theproduct liquid composition comprising the nanoparticle comprising the atleast one long-chain RNA and the at least one cationic or polycationiccompound.
 23. The method according to claim 22, wherein step (e)comprises a drying step.
 24. The method according to claim 21, whereinthe product liquid composition comprising the nanoparticle comprisingthe at least one long-chain RNA and the at least one cationic orpolycationic compound further comprises a pharmaceutically acceptablecarrier.
 25. The method of claim 17, wherein step (c) comprisesintroducing the first liquid composition and the second liquidcomposition into the at least one reactor with a combined flow rate ofat least 250 ml/min.
 26. The method of claim 25, wherein thenanoparticles comprising the at least one long-chain RNA and the atleast one cationic or polycationic compound have a particle size of 50to 200 nm and a polydispersity index (PDI) of 0.4 or below.
 27. Themethod of claim 26, wherein then nanoparticles comprising the at leastone long-chain RNA and the at least one cationic or polycationiccompound have a particle size of 50 to 150 nm.
 28. The method of claim27, wherein then nanoparticles comprising the at least one long-chainRNA and the at least one cationic or polycationic compound have a PDI offrom 0.10 to 0.30.
 29. The method of claim 28, wherein thennanoparticles comprising the at least one long-chain RNA and the atleast one cationic or polycationic compound have a PDI of 0.2 or below.30. The method of claim 28, wherein the reactor comprises at least oneT-piece adapter.
 31. The method of claim 28, wherein the at least onereactor has a volume in a range from 0.1 ml to 10 L.
 32. The method ofclaim 31, wherein the at least one reactor has a volume in a range from1 ml to 5 L.
 33. The method of claim 31, wherein introducing the firstliquid composition and the second liquid composition comprises pumpingthe first liquid composition and the second liquid composition from afirst reservoir and a second reservoir that are each maintained at apredetermined temperature and pressure.
 34. The method of claim 17,wherein step (c) comprises introducing the first liquid composition andthe second liquid composition into the at least one reactor with acombined flow rate of 100-800 ml/min.
 35. The method according to claim1, wherein the first liquid composition and/or the second liquidcomposition further comprise at least one compound selected from a saltor a lyoprotectant.